Polypeptides for a ketoreductase-mediated stereoselective route to alpha chloroalcohols

ABSTRACT

The present disclosure relates to engineered ketoreductase polypeptides and uses thereof for the preparation of α chloroalcohols from α chloroketones. Also provided are polynucleotides encoding the engineered ketoreductase polypeptides and host cells capable of expressing the engineered ketoreductase polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a national stage application filed under 35USC §371 and claims priority of the international applicationPCT/US2010/039511, filed Jun. 22, 2010, and U.S. provisional patentapplications 61/303,057, filed Feb. 10, 2010, and 61/219,162, filed Jun.22, 2009, each of which is hereby incorporated by reference herein.

1. TECHNICAL FIELD

The present disclosure relates to engineered polypeptides and usesthereof for the preparation of α-chloroalcohols from α-chloroketones.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing concurrently submitted electronically under 37C.F.R. §1.821 via EFS-Web in a computer readable form (CRF) as file nameCX2-012WO1_ST25.txt is herein incorporated by reference. The electroniccopy of the Sequence Listing was created on Jun. 22, 2010, with a filesize of 143 kilobytes.

3. BACKGROUND

The stereoselective reduction of an α-halo-ketone to its correspondingchiral halo-alcohol is a transformation found in many useful syntheticroutes. For example, a synthetic route to the antiviral compound,atazanavir, involves the reduction of a Boc-chloro-ketone derived fromL-phenylalanine to the corresponding chiral Boc-(S)-chloro-alcohol.Standard chemical techniques for carrying out this transformation resultin diastereomeric mixtures of the desired intermediate that requirefurther resolution, increasing cost and lowering efficiency in theproduction of atazanavir. Accordingly, processes and compositionscapable of more efficient stereoselective reductions of α-halo-ketonesto chiral halo-alcohols would be desirable.

Certain enzymes belonging to the ketoreductase (KRED) or carbonylreductase class (EC1.1.1.184) have been found to be useful for thestereoselective conversion of pro-stereoisomeric aldehyde or ketonesubstrates to the corresponding chiral alcohol products. KREDs typicallyconvert a ketone or aldehyde substrate to the corresponding alcoholproduct, but may also catalyze the reverse reaction, oxidation of analcohol substrate to the corresponding ketone/aldehyde product. Thereduction of ketones and aldehydes and the oxidation of alcohols byenzymes such as KRED requires a co-factor, most commonly reducednicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adeninedinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide(NAD) or nicotinamide adenine dinucleotide phosphate (NADP) for theoxidation reaction. NADH and NADPH serve as electron donors, while NADand NADP serve as electron acceptors.

KREDs are increasingly being used for the stereoselective conversion ofketones and aldehydes to chiral alcohols compounds used in theproduction of key pharmaceutical compounds. Examples using KREDs togenerate useful chemical compounds include asymmetric reduction of4-chloroacetoacetate esters (Zhou, J. Am. Chem. Soc. 1983 105:5925-5926;Santaniello, J. Chem. Res. (S) 1984:132-133; U.S. Pat. No. 5,559,030;U.S. Pat. No. 5,700,670 and U.S. Pat. No. 5,891,685), reduction ofdioxocarboxylic acids (e.g., U.S. Pat. No. 6,399,339), reduction oftert-butyl (S)chloro-5-hydroxy-3-oxohexanoate (e.g., U.S. Pat. No.6,645,746 and WO 01/40450), reduction of pyrrolotriazine-based compounds(e.g., U.S. application No. 2006/0286646); reduction of substitutedacetophenones (e.g., U.S. Pat. No. 6,800,477); and reduction ofketothiolanes (WO 2005/054491). In another approach, as demonstratedherein, the ketoreduction can be carried out in the presence of analcohol, such as isopropanol, to provide a substrate for the reversereaction (alcohol dehydrogenation). In this manner, the NADH/NADPHconsumed in the ketoreduction reaction is regenerated by the reverse,oxidative reaction.

U.S. Pat. No. 7,083,973 discloses a stereoselective process for thepreparation of (3S,2R)-1-halo-2-hydroxy-3-(protected)amino-4-substitutedbutanes by the reduction of the corresponding keto group containingcompounds using certain species of Rhodococcus and Brevibacterium. The'973 patent discloses that only selected species of Rhodococcus andBrevibacterium catalyze the reduction to form the desired(3S,2R)-1-halo-2-hydroxy-3-(protected)amino-4-substituted butanes inhigh quantitative and enantiomeric yield. The '973 patent discloses that10 mL of cell extract from 150 g of Rhodococcus erythropolis ATCC 4277cells loaded with 10 mg of(1S)-[N-(1-benzyl-2-oxo-3-chloro)propyl]carbamic acid t-butyl estersubstrate, glucose dehydrogenase (35 units), 0.7 mM NAD⁺ and 200 mg ofglucose (reaction carried out at pH 6.0, 150 RPM agitation and 30° C.)results in (1S,2R)-[N-(1-benzyl-2-hydroxy-3-chloro)propyl]carbamic acidt-butyl ester product in 95% yield with >98% diastereomeric purity.

Accordingly, isolated KRED polypeptides capable of stereoselectiveconversion of α-halo-ketones to chiral halo-alcohols in high-yield andhigh diastereomeric purity would be desirable. Also, improved processesfor using KRED polypeptides to carry out large-scale preparation ofchiral halo-alcohols would be desirable.

4. SUMMARY

The present disclosure provides ketoreductase polypeptides capable ofstereoselectively converting an α-halo-ketone to a chiral halo-alcohol,and methods for using these polypeptides in synthetic processes formaking chemical compounds such as intermediates in the production ofactive pharmaceutical ingredients, such as the antiretroviral drug,atazanavir.

In certain embodiments, the disclosure provides ketoreductasepolypeptides capable of converting N-protected(S)-3-amino-1-chloro-4-phenylbutan-2-one compounds of Formula (I) (inwhich R¹ is a protecting group) to the corresponding stereoisomericalcohol N-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol productof Formula (II), as depicted in Scheme 1 below:

In particular embodiments, the present disclosure provides ketoreductasepolypeptides capable of converting chloroketone compound (1)((S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate) to thecorresponding alcohol, compound (2) (tert-butyl(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate), as depicted inScheme 2 below:

In certain embodiments, the present disclosure provides ketoreductasepolypeptides capable of converting a reaction mixture comprising aninitial concentration of at least 10 g/L compound (1) to compound (2)with a conversion rate of at least 70% in 24 hours. In certainembodiments, the concentration of polypeptide capable of carrying outthis conversion is 5 g/L, 2 g/L, 1 g/L, or less. In certain embodiments,the polypeptides are capable of a conversion rate of at least 80%, 85%,90%, 95%, 98%, 99%, or more in 24 hours, or even less time. In certainembodiments, the polypeptides are capable of converting compound (1) tocompound (2) in a diastereomeric excess of greater than about 95%,greater than about 97%, or greater than about 99%. In certainembodiments, the polypeptides are capable of the above conversion rateswith a reaction mixture comprising an initial concentration of compound(1) of at least 20 g/L, 40 g/L, 60 g/L, 80 g/L, 100 g/L, 150 g/L, 200g/L, or even more.

In certain embodiments, the present disclosure provides a method forconverting a compound of Formula (I) to a compound of Formula (III) (seeScheme 3), wherein R′ is as described above, comprising converting thecompound of Formula (I) to a compound of Formula (II) using aketoreductase of the present disclosure and then contacting the compoundof Formula (II) with base to provide the compound of Formula (III).

In certain embodiments, the method further comprises extracting thereaction mixture comprising the compound of Formula (II) into an organicsolvent extract and contacting the extract with base. In certainembodiments, the method further comprises exchanging the organic solventextract with a crystallization solvent and crystallizing the compound ofFormula (III). In certain embodiments, the step of contacting thecompound of Formula (II) with base is carried out without firstpurifying and/or isolating the compound of Formula (II).

In a specific embodiment, therefore, the present disclosure provides amethod for converting compound (1) to compound (3)(tert-butyl(S)-1-((R)-oxiran-2-yl)-2-phenylethylcarbamate) comprisingconverting compound (1) to compound (2) using a ketoreductase of thepresent disclosure and then contacting compound (2) with base to providecompound (3) (see Scheme 4).

In certain embodiments, the method further comprises extracting thereaction mixture comprising the compound (2) into an organic solventextract and contacting the extract with base. In certain embodiments,the method further comprises exchanging the organic solvent extract witha crystallization solvent and crystallizing the compound (3). In certainembodiments, the step of contacting the compound (2) with base iscarried out without first purifying and/or isolating compound (2).

In some embodiments, the method for reducing or converting thesubstrate, N-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one (e.g.compound (1)) to its corresponding stereoisomeric alcohol product,N-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol (e.g., compound(2)), comprises contacting or incubating the substrate with at least oneof the ketoreductase polypeptides disclosed herein under reactionconditions suitable for reducing or converting the substrate to theproduct.

In some embodiments of the above methods, the substrate is reduced tothe product in greater than about 95%, greater than about 97%, orgreater than about 99% diastereomeric excess, wherein the ketoreductasepolypeptide comprises a sequence that corresponds to SEQ ID NO: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56 and 58.

In certain embodiments of the above methods, at least about 95% of thesubstrate is converted to the product in less than about 24 hours whencarried out with greater than about 100 g/L of substrate and less thanabout 5 g/L of the polypeptide. In certain embodiments, the polypeptidecapable of carrying out the method comprises an amino acid sequencecorresponding to SEQ ID NO: 6, 50, 52, and 56. In some embodiments ofthe above methods, at least about 95% of the substrate is converted tothe product in less than about 30 hours when carried out with greaterthan about 150 g/L of substrate and less than about 1 g/L of thepolypeptide, wherein the polypeptide comprises an amino acid sequencecorresponding to SEQ ID NO: 6, 50, 52, and 56.

In one aspect, the ketoreductase polypeptides described herein haveamino acid sequences with one or more amino acid differences as comparedto a wild-type ketoreductase or as compared to an engineeredketoreductase. The one or more amino acid differences result in at leastone improved property of the enzyme for a defined substrate. Generally,the ketoreductase polypeptides described herein are engineeredketoreductase polypeptides having one or more improved properties ascompared to the naturally-occurring wild-type ketoreductase enzymesobtained from Novosphingobium aromaticivorans (“N. aromaticivorans”; SEQID NO:2). Improvements in an enzyme property of the engineeredketoreductase polypeptides include increases in enzyme activity,stereoselectivity, sterospecificity, thermostability, solvent stability,tolerance to increased levels of substrate, and tolerance to increasedlevels product.

In some embodiments, the ketoreductase polypeptides of the invention areimproved as compared to SEQ ID NO:2 with respect to their rate ofenzymatic activity, i.e., the conversion rate for reducing N-protected(S)-3-amino-1-chloro-4-phenylbutan-2-one (“the substrate”) (e.g.compound (1) where the protecting group is a BOC moiety) to N-protected(2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol (“the product”) (e.g.,compound (2)). In some embodiments, the engineered ketoreductasepolypeptides are capable of converting the substrate to the product at aconversion rate that is at least at least 1.1-times, 1.2-times,1.3-times, 1.5-times, 2-times, 3-times, or more than 3-times the rateexhibited by the enzyme of SEQ ID NO: 2 under comparable assayconditions.

In some embodiments, such ketoreductase polypeptides are also capable ofconverting the N-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one(“the substrate”) (e.g., compound (1) where the protecting group is aBOC moiety) to N-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol(“the product”) (e.g. compound (2)), with a percent diastereomericexcess of at least about 95%. In some embodiments, such ketoreductasepolypeptides are also capable of converting the substrate to the productwith a percent diastereomeric excess of at least about 97%. In someembodiments, such ketoreductase polypeptides are also capable ofconverting the substrate to the product with a percent diastereomericexcess of at least about 99%. Exemplary polypeptides with suchproperties include, but are not limited to, polypeptides whichcomprising amino acid sequences corresponding to SEQ ID NO: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56 and 58.

In some embodiments, the ketoreductase polypeptide is capable ofconverting N-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one (“thesubstrate”) (e.g. compound (1)) where the protecting group is a BOCmoiety) to N-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol (“theproduct”) (e.g. compound (2)), with a percent diastereomeric excess ofat least about 99% and at a conversion rate that is at least about 1.2times or more improved over the polypeptide of SEQ ID NO:2. Exemplarypolypeptides with such properties include, but are not limited to,polypeptides which comprise an amino acid sequence corresponding to SEQID NO: 4, 6, 14, 16, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 50, 52,54, and 56.

In some embodiments, the ketoreductase polypeptide is capable ofconverting N-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one (“thesubstrate”) (e.g., compound (1) where the protecting group is a BOCmoiety) to N-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol (“theproduct”) (e.g. compound (2)), with a percent diastereomeric excess ofat least about 99% and at a conversion rate that is at least about 1.5times or more improved over the polypeptide of SEQ ID NO:2. Exemplarypolypeptides with such properties include, but are not limited to,polypeptides which comprise an amino acid sequence corresponding to SEQID NO: 6, 18, 22, 30, 38, 40, 50, 52, 54, and 56.

In some embodiments, the ketoreductase polypeptide is capable ofconverting N-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one (“thesubstrate”) (e.g. compound (1) where the protecting group is a BOCmoiety) to N-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol (“theproduct”) (e.g. compound (2)), with a percent diastereomeric excess ofat least about 99% and at a conversion rate that is more than 3 timesimproved over the polypeptide of SEQ ID NO:2. Exemplary polypeptideswith such properties include, but are not limited to, polypeptides whichcomprise an amino acid sequence corresponding to SEQ ID NO: 6, 50, 52,and 56.

In some embodiments, the ketoreductase polypeptide is capable ofconverting at least about 95% of the substrate to the product in lessthan about 24 hours when carried out with greater than about 100 g/L ofsubstrate and less than about 5 g/L of the polypeptide. Exemplarypolypeptides that have this capability include, but are not limited to,polypeptides which comprise amino acid sequences corresponding to SEQ IDNO: 6, 50, 52, and 56.

In some embodiments, the ketoreductase polypeptide is highlystereoselective, wherein the polypeptide can reduce the substrate to theproduct in greater than about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8% or 99.9% diastereomeric excess. Exemplaryketoreductase polypeptides with high stereoselectivity include, but arenot limited to, the polypeptides comprising the amino acid sequencescorresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 and 58.

In some embodiments, the ketoreductase polypeptide has the improvedproperty of increased activity with a secondary alcohol for cofactorregeneration. In some embodiments, the ketoreductase polypeptideoxidizes isopropanol (IPA) to acetone with an activity at least 2-fold,2.5-fold, 5-fold, 10-fold, 15-fold, or even greater, relative to thereference polypeptide of SEQ ID NO: 2. Exemplary ketoreductasepolypeptides exhibiting the improved property of increased activity withIPA include, but are not limited to, the polypeptides comprising theamino acid sequence corresponding to SEQ ID NO: 6, 56, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, and 80.

In certain embodiments, a ketoreductase polypeptide of the presentdisclosure is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thereference sequence of SEQ ID NO:2, and has, at the positioncorresponding to the indicated position of SEQ ID NO:2, at least one ofthe following amino acid differences: the amino acid at position 2 is analiphatic or nonpolar amino acid selected from among alanine, leucine,valine, isoleucine, glycine and methionine; the amino acid at position28 is an aliphatic or nonpolar amino acid selected from among alanine,leucine, valine, isoleucine, glycine and methionine; the amino acid atposition 34 is a polar amino acid selected from among asparagine,glutamine, serine, and threonine; the amino acid at position 47 is analiphatic or nonpolar amino acid selected from among alanine, leucine,valine, isoleucine, glycine and methionine; the amino acid at position50 is a basic amino acid selected from lysine and arginine; the aminoacid at position 81 is a polar amino acid selected from amongasparagine, glutamine, serine, and threonine; the amino acid at position90 is an aliphatic or nonpolar amino acid selected from among alanine,leucine, valine, isoleucine, glycine and methionine; the amino acid atposition 91 is either an aliphatic or nonpolar amino acid selected fromamong alanine, leucine, valine, isoleucine, glycine and methionine, orthe amino acid at position 91 is an aromatic amino acid selected fromamong tyrosine, tryptophan, and phenylalanine, or the amino acid atposition 91 basic amino acid selected from among lysine and arginine;the amino acid at position 94 is the basic amino acid, arginine; theamino acid at position 112 is an aromatic amino acid selected from amongtyrosine, tryptophan, and phenylalanine; the amino acid at position 117is an acidic amino acid selected from among aspartic acid and glutamicacid; the amino acid at position 143 is a basic amino acid selected fromamong lysine and arginine; the amino acid at position 144 is either acysteine, or the amino acid at position 144 is a polar amino acidselected from among asparagine, glutamine, serine, and threonine; theamino acid at position 145 is either a nonpolar amino acid selected fromamong alanine, leucine, valine, isoleucine, and methionine or analiphatic amino acid selected from among alanine, leucine, valine,isoleucine; the amino acid at position 148 is a constrained amino acidselected from among proline and histidine; the amino acid at position150 is either a nonpolar or aliphatic amino acid selected from amongleucine, valine, isoleucine, glycine and methionine, or the amino acidat position 150 is a polar amino acid selected from among asparagine,glutamine, serine, and threonine, or the amino acid at position 150 isan aromatic amino acid selected from among tyrosine, tryptophan, andphenylalanine; the amino acid at position 152 is a nonpolar or aliphaticamino acid selected from among alanine, leucine, valine, isoleucine,glycine and methionine; the amino acid at position 153 is either anonpolar or aliphatic amino acid selected from among alanine, leucine,valine, isoleucine, glycine and methionine, or a constrained amino acidselected from among histidine and proline; the amino acid at position158 is a polar amino acid selected from among asparagine, glutamine, andserine; the amino acid at position 190 is either a nonpolar or analiphatic amino acid selected from among alanine, valine, leucine,isoleucine, glycine, and methionine, or the amino acid at position 190is a polar amino acid selected from among asparagine, glutamine, andserine, or the amino acid at position 190 is a proline; the amino acidat position 198 is a polar amino acid selected from among asparagine,glutamine, and threonine; the amino acid at position 199 is either analiphatic or nonpolar amino acid selected from among alanine, leucine,valine, glycine, and methionine, or a polar amino acid selected fromamong asparagine, glutamine, serine, and threonine; the amino acid atposition 200 is a nonpolar amino acid selected from among alanine,leucine, valine, isoleucine, and glycine; the amino acid at position 204is an aromatic amino acid selected from among tyrosine, tryptophan, andphenylalanine; the amino acid at position 217 is a polar amino acidselected from among asparagine, glutamine, serine and threonine; theamino acid at position 225 is a nonpolar amino acid selected from amongvaline, leucine, glycine, and methionine; the amino acid at position 231is an aromatic amino acid selected from among tyrosine, tryptophan, andphenylalanine; the amino acid at position 232 is a nonpolar amino acidselected from among leucine, isoleucine, valine, glycine, andmethionine; the amino acid at position 233 is a polar amino acidselected from among asparagine, glutamine, serine, and threonine; theamino acid at position 244 is a nonpolar amino acid selected from amongalanine, leucine, isoleucine, valine, glycine, and methionine; the aminoacid at position 260 is an aromatic amino acid selected from amongtyrosine and tryptophan; and the amino acid at position 261 is a polaramino acid selected from among asparagine, glutamine, and threonine.

In certain embodiments, a ketoreductase polypeptide of the presentdisclosure is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thereference sequence of SEQ ID NO:2, and has, as compared to SEQ ID NO:2,at least one amino acid substitution selected from the group consistingof: P2L; V28A; A34S; A47V; E50K; D81N; S90V; I91L; I91W; I91R; I91K;K94R; D112Y; G117D; S143R; V144C; V144T; G145A; G145V; R148H; A150G;A1501; A150S; A150W; F152L; N153G; N153V; N153H; T158S; G190A; G190P;G190Q; G190V; S198N; I199G; I199L; I199M; I199N; M200I; V204F; A217T;I225V; P231F; A232V; E233Q; D244G; F260Y; S261N; and mixtures thereof.

In some embodiments, an improved ketoreductase polypeptide of thedisclosure is based on the sequence of SEQ ID NO:2 and comprises anamino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thereference sequence of SEQ ID NO:2, and, further comprises at least oneamino acid substitution selected from the group consisting of: theproline residue at position 2 is replaced with leucine; the valineresidue at position 28 is replaced with alanine; the alanine residue atposition 34 is replaced with serine; the alanine residue at position 47is replaced with valine; the glutamic acid residue at position 50 isreplaced with lysine; the aspartic acid residue at position 81 isreplaced with asparagine; the serine residue at position 90 is replacedwith valine; the isoleucine residue at position 91 is replaced with anamino acid selected from among leucine, tryptophan, arginine, andlysine; the lysine residue at position 94 is replaced with arginine; theaspartic acid residue at position 112 is replaced with tyrosine; theglycine residue at position 117 is replaced with aspartic acid; theserine residue at position 143 is replaced with arginine; the valineresidue at position 144 is replaced with an amino acid selected fromamong cysteine and threonine; the glycine residue at position 145 isreplaced with an amino acid selected from among alanine and valine; thearginine residue at position 148 is replaced with histidine; the alanineresidue at position 150 is replaced with an amino acid selected fromamong glycine, isoleucine, serine, and tryptophan; the phenylalanineresidue at position 152 is replaced with leucine; the asparagine residueat position 153 is replaced with an amino acid selected from amongglycine, valine, and histidine; the threonine residue at position 158 isreplaced with serine; the glycine residue at position 190 is replacedwith an amino acid selected from among alanine, proline, glutamine, andvaline; the serine residue at position 198 is replaced with asparagine;the isoleucine residue at position 199 is replaced with an amino acidselected from among glycine, methionine, leucine, and asparagine; themethionine residue at position 200 is replaced with isoleucine; thevaline residue at position 204 is replaced with phenylalanine; thealanine residue at position 217 is replaced with threonine; theisoleucine residue at position 225 is replaced with valine; the prolineresidue at position 231 is replaced with phenylalanine; the alanineresidue at position 232 is replaced with valine; the glutamic acidresidue at position 233 is replaced with glutamine; the aspartic acidresidue at position 244 is replaced with glycine; the phenylalanineresidue at position 260 is replaced with tyrosine; and the serineresidue at position 261 is replaced with asparagine.

In some embodiments, the ketoreductase polypeptides can have, inaddition to the above, one or more modifications (i.e., residuedifferences) as compared to the reference amino acid sequence or ascompared to any of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, or 80. These modifications can beamino acid insertions, deletions, substitutions, or any combination ofsuch changes. In some embodiments, the amino acid sequence differencescan comprise non-conservative, conservative, as well as a combination ofnon-conservative and conservative amino acid substitutions. In someembodiments, these ketoreductase polypeptides can have optionally fromabout 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40mutations at other amino acid residues. In some embodiments, the numberof modifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15,16, 18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.

In some embodiments, an improved ketoreductase comprises an amino acidsequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence corresponding to SEQ ID NO: 2, wherein the improvedketoreductase polypeptide amino acid sequence includes any one set ofthe specified amino acid substitution combinations presented in Tables 2or 3. In some embodiments, these ketoreductase polypeptides can havemutations at other amino acid residues.

In another aspect, the present disclosure provides polynucleotidesencoding the ketoreductase polypeptides described herein, andpolynucleotides that hybridize to such polynucleotides under highlystringent conditions. The polynucleotide can include promoters and otherregulatory elements useful for expression of the encoded engineeredketoreductase, and can utilize codons optimized for specific desiredexpression systems.

In some embodiments, the disclosure provides polynucleotides encodingketoreductase polypeptides having at least the following amino acidsequence as compared to the amino acid sequence of SEQ ID NO:2, andfurther comprising at least one acid substitution selected from thegroup consisting of: the proline residue at position 2 is replaced withleucine; the valine residue at position 28 is replaced with alanine; thealanine residue at position 34 is replaced with serine; the alanineresidue at position 47 is replaced with valine; the glutamic acidresidue at position 50 is replaced with lysine; the aspartic acidresidue at position 81 is replaced with asparagine; the serine residueat position 90 is replaced with valine; the isoleucine residue atposition 91 is replaced with an amino acid selected from among leucine,tryptophan, arginine, and lysine; the lysine residue at position 94 isreplaced with arginine; the aspartic acid residue at position 112 isreplaced with tyrosine; the glycine residue at position 117 is replacedwith aspartic acid; the serine residue at position 143 is replaced witharginine; the valine residue at position 144 is replaced with an aminoacid selected from among cysteine and threonine; the glycine residue atposition 145 is replaced with an amino acid selected from among alanineand valine; the arginine residue at position 148 is replaced withhistidine; the alanine residue at position 150 is replaced with an aminoacid selected from among glycine, isoleucine, serine, and tryptophan;the phenylalanine residue at position 152 is replaced with leucine; theasparagine residue at position 153 is replaced with an amino acidselected from among glycine, valine, and histidine; the threonineresidue at position 158 is replaced with serine; the glycine residue atposition 190 is replaced with an amino acid selected from among alanine,proline, glutamine, and valine; the serine residue at position 198 isreplaced with asparagine; the isoleucine residue at position 199 isreplaced with an amino acid selected from among glycine, methionine,leucine, and asparagine; the methionine residue at position 200 isreplaced with isoleucine; the valine residue at position 204 is replacedwith phenylalanine; the alanine residue at position 217 is replaced withthreonine; the isoleucine residue at position 225 is replaced withvaline; the proline residue at position 231 is replaced withphenylalanine; the alanine residue at position 232 is replaced withvaline; the glutamic acid residue at position 233 is replaced withglutamine; the aspartic acid residue at position 244 is replaced withglycine; the phenylalanine residue at position 260 is replaced withtyrosine; and the serine residue at position 261 is replaced withasparagine. Exemplary polynucleotides include, but are not limited to, apolynucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79.

In some embodiments, the present disclosure provides host cellscomprising the polynucleotides encoding the ketoreductase polypeptidesas described herein and/or expression vectors comprising thesepolynucleotides. The host cells may be N. aromaticivorans or they may bea different organism, and as E. coli. The host cells can be used for theexpression and isolation of the engineered ketoreductase enzymesdescribed herein, or, alternatively, they can be used directly for theconversion of the substrate to the stereoisomeric product. Accordingly,in some embodiments, the engineered ketoreductase polypeptides disclosedherein can be prepared by standard methods comprising culturing a hostcell containing an expression vector comprising a polynucleotideencoding the polypeptide and isolating the polypeptide from the hostcell.

Whether carrying out the method with whole cells, cell extracts orpurified ketoreductase enzymes, a single ketoreductase enzyme may beused or, alternatively, mixtures of two or more ketoreductase enzymesmay be used.

5. DETAILED DESCRIPTION

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients, reaction conditions, andso forth used in the specification and claims are to be understood asbeing modified in all instances by the term “about.”

In this disclosure, the use of the singular includes the plural (andvice versa) unless specifically stated otherwise. Also, the use of “or”means “and/or” unless stated otherwise. Similarly, “comprise,”“comprises,” “comprising” “include,” “includes,” and “including” areinterchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

It is to be understood that both the foregoing general description,including the drawings, and the following detailed description areexemplary and explanatory only and are not restrictive of thisdisclosure.

The section headings used herein are for organizational purposes onlyand not to be construed as limiting the subject matter described.

The present disclosure is directed to biocatalytic processes in whichα-chloroketones are contacted with a ketoreductase enzyme and therebyconcerted to the corresponding α-chloro alcohol. The present disclosuretherefore provides ketoreductase enzymes capable of convertingN-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one (“the substrate”)(e.g. compound (1) where the protecting group is a BOC moiety) to thecorresponding stereoisomeric alcohol product N-protected(2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol (“the product”) (e.g.compound (2)). The present disclosure further comprises a method forconverting that alcohol product, an N-protected(2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol (e.g. compound (2)), to thecorresponding stereoisomeric epoxide, N-protected(S)-1-((R)-oxiran-2-yl)-2-phenylethylcarbamate), e.g. compound (3)(tert-butyl(S)-1-((R)-oxiran-2-yl)-2-phenylethylcarbamate; where theprotecting group is a BOC moiety).

5.1. Definitions

As used herein, the following terms are intended to have the followingmeanings:

The term “protecting group” refers to a group of atoms that whenattached to a reactive functional group in a molecule, mask, reduce orprevent the reactivity of the functional group. Typically, a protectinggroup may be selectively removed as desired during the course of asynthesis.

“Nitrogen protecting group” (or “N-protecting group”) means asubstituent commonly employed to block or protect a nitrogenfunctionality while reacting other functional groups on a compound.Examples of such nitrogen-protecting groups include the formyl group,the trityl group, the methoxytrityl group, the tosyl group, thephthalimido group, the acetyl group, the trichloroacetyl group, thechloroacetyl, bromoacetyl, and iodoacetyl groups, benzyloxycarbonyl(Cbz), 9-fluorenylmethoxycarbonyl (FMOC), 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), trihaloacetyl, benzyl, benzoyl, andnitrophenylacetyl and the like. Further examples of protecting groupsuseful with the embodiments of the present disclosure can be found in P.G. M. Wuts and T. W. Greene, “Greene's Protective Groups in OrganicSynthesis—Fourth Edition,” John Wiley and Sons, New York, N.Y., 2007,Chapter 7 (“Greene”).

“Stereoisomer,” “stereoisomeric form,” and the like are general termsfor all isomers of individual molecules that differ only in theorientation of their atoms in space. In includes enantiomers and isomersof compounds with more than one chiral center that are not mirror imagesof one another (“diastereomers”).

“Chiral center” refers to a carbon atom to which four different groupsare attached.

“Enantiomer” or “enantiomeric” refers to a molecule that isnonsuperimposable on its mirror image and hence optically active wherethe enantiomer rotates the plane of polarized light in one direction andits mirror image rotates the plane of polarized light in the oppositedirection.

“Enantiomeric excess,” “(ee),” “diastereomeric excess,” “(de),” meansthat one enantiomer or diastereomer is present more than the other in achemical substance. This difference is defined as the absolutedifference between the mole fractions of each enantiomer:ee=|(F+)−(F−)|, where (F+)+(F−)=1. Thus, (ee) and (de) can expressed asa percent enantiomeric or diastereomeric excess.

The term “racemic” refers to a mixture of equal molar amounts of twoenantiomers of a compound, which mixture is optically inactive.

As used herein, a composition is “enriched” in a particular chiralcompound, enantiomer, or diastereomer will typically comprise at leastabout 60%, 70%, 80%, 90%, or even more of that particular chiralcompound, enantiomer, or diastereomer. The amount of enrichment can bedetermined using conventional analytical methods routinely used by thoseof ordinary skill in the art, including but not limited to, NMRspectroscopy in the presence of chiral shift reagents, gaschromatographic analysis using chiral columns, and high pressure liquidchromatographic analysis using chiral columns. In some embodiments asingle chiral compound, enantiomer, or diastereomer will besubstantially free of other corresponding chiral compound, enantiomer,or diastereomers. By “substantially free” is meant that the compositioncomprises less than about 10% of the specified undesired chiralcompound, enantiomer, or diastereomer as established using conventionalanalytical methods routinely used by those of ordinary skill in the art,such as the methods noted above. In some embodiments, the amount ofundesired chiral compound, enantiomer, or diastereomer may be less thanabout 10%, for example, less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1% or even less. Chirally enantiomerically, or diastereomericallyenriched compositions that contain at least about 95% of a specifiedchiral compound, enantiomer, or diastereomer are referred to herein as“substantially chirally pure,” “substantially enantiomerically pure” and“substantially diastereomerically pure,” respectively. Compositions thatcontain at least about 99% of a specified chiral compound, enantiomer,or diastereomer are referred to herein as “chirally pure,”“enantiomerically pure,” and “diastereomerically pure,” respectively.

“Ketoreductase” and “KRED” are used interchangeably herein to refer to apolypeptide having an enzymatic capability of reducing a carbonyl groupto its corresponding alcohol. In specific embodiments, the ketoreductasepolypeptides of the invention are capable of stereoselectively reducing(1) ((S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate) to thecorresponding alcohol, compound (2) (tert-butyl(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate). The polypeptidetypically utilizes a cofactor reduced nicotinamide adenine dinucleotide(NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) asthe reducing agent. Ketoreductases as used herein include naturallyoccurring (wild type) ketoreductases as well as non-naturally occurringengineered polypeptides generated by human manipulation.

“Engineered ketoreductase polypeptide” as used herein refers to anketoreductase polypeptide having a variant sequence generated by humanmanipulation (e.g., a sequence generated by directed evolution of anaturally occurring parent enzyme or directed evolution of a variantpreviously derived from a naturally occurring enzyme).

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Polynucleotides” or “oligonucleotides” refer to nucleobase polymers oroligomers in which the nucleobases are connected by sugar phosphatelinkages (sugar-phosphate backbone). Nucleobase or base includenaturally occurring and synthetic heterocyclic moieties commonly knownto those who utilize nucleic acid or polynucleotide technology orutilize polyamide or peptide nucleic acid technology to thereby generatepolymers that can hybridize to polynucleotides in a sequence-specificmanner. Non-limiting examples of nucleobases include: adenine, cytosine,guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil,5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine,2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine),hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). Exemplary poly- and oligonucleotides includepolymers of 2′ deoxyribonucleotides (DNA) and polymers ofribonucleotides (RNA). A polynucleotide may be composed entirely ofribonucleotides, entirely of 2′ deoxyribonucleotides or combinationsthereof.

“Coding sequence” refers to that portion of a polynucleotide (e.g., agene) that encodes an amino acid sequence of a polypeptide (e.g., aprotein).

“Percentage of sequence identity,” “percent identity,” and “percentidentical” are used herein to refer to comparisons betweenpolynucleotide sequences or polypeptide sequences, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which either the identical nucleic acid base or amino acidresidue occurs in both sequences or a nucleic acid base or amino acidresidue is aligned with a gap to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Determination of optimalalignment and percent sequence identity is performed using the BLAST andBLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as, the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915).

Other algorithms are available that function similarly to BLAST inproviding percent identity for two sequences. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe GCG Wisconsin Software Package), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)(Ausubel)). Additionally, determination of sequence alignment andpercent sequence identity can employ the BESTFIT or GAP programs in theGCG Wisconsin Software package (Accelrys, Madison Wis.), using defaultparameters provided.

“Reference sequence” refers to a defined sequence to which an alteredsequence is compared. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a comparison window to identify and compare local regions ofsequence similarity.

The term “reference sequence” is not intended to be limited to wild-typesequences, and can include engineered or altered sequences. For example,in some embodiments, a “reference sequence” can be a previouslyengineered or altered amino acid sequence. For instance, a “referencesequence based on SEQ ID NO: 2 having a glycine residue at positionX315” refers to a reference sequence corresponding to SEQ ID NO:2 with aglycine residue at X315 (whereas the un-altered version of SEQ ID NO:2has glutamate at X315).

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent sequence identity, at least 89 percent sequence identity, atleast 95 percent sequence identity, and even at least 99 percentsequence identity as compared to a reference sequence over a comparisonwindow of at least 20 residue positions, frequently over a window of atleast 30-50 residues, wherein the percentage of sequence identity iscalculated by comparing the reference sequence to a sequence thatincludes deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. In specificembodiments applied to polypeptides, the term “substantial identity”means that two polypeptide sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 89 percent sequenceidentity, at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

“Corresponding to,” “reference to,” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredketoreductase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned.

“Derived from” as used herein in the context of engineered enzymesidentifies the originating enzyme, and/or the gene encoding such enzyme,upon which the engineering was based. For example, the engineeredketoreductase enzyme having variant polypeptide sequence SEQ ID NO: 6was obtained by artificially mutating, over multiple generations thepolynucleotide encoding the wild-type ketoreductase enzyme of SEQ IDNO:2. Thus, this engineered ketoreductase enzyme is “derived from” thewild-type ketoreductase of SEQ ID NO: 2.

“Stereoselectivity” or “stereospecificity” refer to the preferentialformation in a chemical or enzymatic reaction of one stereoisomer overanother. Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity sometimes is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” as used herein refers to a ketoreductasepolypeptide that is capable of converting or reducing a substrate to thecorresponding product (e.g., compound (1) to compound (2)) with at leastabout 99% stereomeric excess.

“Improved enzyme property” refers to any enzyme property made better ormore desirable for a particular purpose as compared to that propertyfound in a reference enzyme. For the engineered ketoreductasepolypeptides described herein, the comparison is generally made to thewild-type ketoreductase enzyme, although in some embodiments, thereference ketoreductase can be another improved engineeredketoreductase. Enzyme properties for which improvement is desirableinclude, but are not limited to, enzymatic activity (which can beexpressed in terms of percent conversion of the substrate in a period oftime), thermal stability, pH stability or activity profile, cofactorrequirements, refractoriness to inhibitors (e.g., product inhibition),stereospecificity, and stereoselectivity (including enantioselectivity).

“Increased enzymatic activity” or “increased activity” or “increasedconversion rate” refers to an improved property of an engineered enzyme,which can be represented by an increase in specific activity (e.g.,product produced/time/weight protein) or an increase in conversion rateof the substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of transaminase) as compared to a reference enzyme.Exemplary methods to determine enzyme activity and conversion rate areprovided in the Examples. Any property relating to enzyme activity maybe affected, including the classical enzyme properties of K_(m), V_(max)or k_(cat), changes of which can lead to increased enzymatic activity.Improvements in enzyme activity can be from about 100% improved over theenzymatic activity of the corresponding wild-type ketoreductase enzyme,to as much as 200%, 500%, 1000%, or more over the enzymatic activity ofthe naturally occurring ketoreductase or another engineeredketoreductase from which the ketoreductase polypeptides were derived. Inspecific embodiments, the engineered ketoreductase enzyme exhibitsimproved enzymatic activity in the range of a 100% to 200%, 200% to1000% or more than a 1500% improvement over that of the parent,wild-type or other reference ketoreductase enzyme. It is understood bythe skilled artisan that the activity of any enzyme is diffusion limitedsuch that the catalytic turnover rate cannot exceed the diffusion rateof the substrate, including any required cofactors. The theoreticalmaximum of the diffusion limit, or k_(cat)/K_(m), is generally about 10⁸to 10⁹ (M⁻¹s⁻¹). Hence, any improvements in the enzyme activity of theketoreductase will have an upper limit related to the diffusion rate ofthe substrates acted on by the ketoreductase enzyme. Ketoreductaseactivity can be measured by any one of standard assays used formeasuring ketoreductase, such as the assay described in Example 7.Comparisons of enzyme activities or conversion rates are made using adefined preparation of enzyme, a defined assay under a set condition,and one or more defined substrates, as further described in detailherein. Generally, when lysates are compared, the numbers of cellsand/or the amount of protein assayed are determined as well as use ofidentical expression systems and identical host cells to minimizevariations in amount of enzyme produced by the host cells and present inthe lysates.

“Conversion” refers to the enzymatic transformation of a substrate tothe corresponding product. “Percent conversion” refers to the percent ofthe substrate that is converted to the product within a period of timeunder specified conditions. Thus, for example, the “activity” or“conversion rate” of a ketoreductase polypeptide can be expressed as“percent conversion” of the substrate to the product.

“Thermostable” or “thermal stable” are used interchangeably to refer toa polypeptide that is resistant to inactivation when exposed to a set oftemperature conditions (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme, thus retaining a certainlevel of residual activity (more than 60% to 80% for example) afterexposure to elevated temperatures.

“Solvent stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to varying concentrations(e.g., 5-99%) of solvent, (e.g., isopropyl alcohol, dimethylsulfoxide,tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, acetonitrile, etc.) for a periodof time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

“pH stable” refers to a polypeptide that maintains similar activity(more than e.g. 60% to 80%) after exposure to high or low pH (e.g. 8 to12 or 4.5-6) for a period of time (e.g. 0.5-24 hrs) compared to theuntreated enzyme.

“Thermo- and solvent stable” refers to a polypeptide that is boththermostable and solvent stable.

“Amino acid” or “residue” as used in context of the polypeptidesdisclosed herein refers to the specific monomer at a sequence position(e.g., E315 indicates that the “amino acid” or “residue” at position 315of SEQ ID NO: 2 is a glutamate).

“Hydrophilic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-Arg (R) and L-Lys (K).

“Polar Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic Amino Acid or Residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L His (H) it is sometimesclassified as a basic residue, or as an aromatic residue as its sidechain includes a heteroaromatic ring, herein histidine is classified asa hydrophilic residue or as a “constrained residue” (see below).

“Constrained amino acid or residue” refers to an amino acid or residuethat has a constrained geometry. Herein, constrained residues includeL-pro (P) and L-his (H). Histidine has a constrained geometry because ithas a relatively small imidazole ring. Proline has a constrainedgeometry because it also has a five membered ring.

“Non-polar Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

“Cysteine”. The amino acid L-Cys (C) is unusual in that it can formdisulfide bridges with other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with SH containing side chains) to exist in apeptide in either the reduced free SH or oxidized disulfide-bridged formaffects whether L-Cys (C) contributes net hydrophobic or hydrophiliccharacter to a peptide. While L-Cys (C) exhibits a hydrophobicity of0.29 according to the normalized consensus scale of Eisenberg (Eisenberget al., 1984, supra), it is to be understood that for purposes of thepresent disclosure L-Cys (C) is categorized into its own unique group.

“Small Amino Acid or Residue” refers to an amino acid or residue havinga side chain that is composed of a total three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include L-Ala(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp(D).

“Hydroxyl-containing Amino Acid or Residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S), L-Thr (T) and L-Tyr(Y).

“Amino acid difference” or “residue difference” refers to a change inthe residue at a specified position of a polypeptide sequence whencompared to a reference sequence. For example, a residue difference atposition I199, where the reference sequence has a isoleucine, refers toa change of the residue at position 199 to any residue other thanisoleucine. As disclosed herein, an engineered ketoreductase enzyme caninclude one or more residue differences relative to a referencesequence, where multiple residue differences typically are indicated bya list of the specified positions where changes are made relative to thereference sequence (e.g., “one or more residue differences as comparedto SEQ ID NO: 2 at the following residue positions: 2, 28, 34, 47, 50,81, 90, 91, 94, 112, 117, 143, 144, 145, 150, 152, 153, 158, 190, 198,199, 200, 204, 217, 225, 231, 232, 233, 244, 260, and 261.”)

A “conservative” amino acid substitution (or mutation) refers to thesubstitution of a residue with a residue having a similar side chain,and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. However, as used herein, in some embodiments, conservativemutations do not include substitutions from a hydrophilic tohydrophilic, hydrophobic to hydrophobic, hydroxyl-containing tohydroxyl-containing, or small to small residue, if the conservativemutation can instead be a substitution from an aliphatic to analiphatic, non-polar to non-polar, polar to polar, acidic to acidic,basic to basic, aromatic to aromatic, or constrained to constrainedresidue. Further, as used herein, A, V, L, or I can be conservativelymutated to either another aliphatic residue or to another non-polarresidue. Table 1 below shows exemplary conservative substitutions.

TABLE 1 Conservative Substitutions Residue Possible ConservativeMutations A, L, V, I Other aliphatic (A, L, V, I) Other non-polar (A, L,V, I, G, M) G, M Other non-polar (A, L, V, I, G, M) D, E Other acidic(D, E) K, R Other basic (K, R) P, H Other constrained (P, H) N, Q, S, TOther polar (N, Q, S, T) Y, W, F Other aromatic (Y, W, F) C None

“Non-conservative substitution” refers to substitution or mutation of anamino acid in the polypeptide with an amino acid with significantlydiffering side chain properties. Non-conservative substitutions may useamino acids between, rather than within, the defined groups listedabove. In one embodiment, a non-conservative mutation affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 3 ormore amino acids, 4 or more amino acids, 5 or more amino acids, 6 ormore amino acids, 7 or more amino acids, 8 or more amino acids, 10 ormore amino acids, 12 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered ketoreductase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered ketoreductase enzymes comprise insertions of oneor more amino acids to the naturally occurring ketoreductase polypeptideas well as insertions of one or more amino acids to other engineeredketoreductase polypeptides. Insertions can be in the internal portionsof the polypeptide, or to the carboxy or amino terminus. Insertions asused herein include fusion proteins as is known in the art. Theinsertion can be a contiguous segment of amino acids or separated by oneor more of the amino acids in the naturally occurring polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of a full-length ketoreductase polypeptide.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved ketoreductase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved ketoreductase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure ketoreductase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedimproved ketoreductases polypeptide is a substantially pure polypeptidecomposition.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(Tm) of the hybrids. In general, the stability of a hybrid is a functionof ion strength, temperature, G/C content, and the presence ofchaotropic agents. The Tm values for polynucleotides can be calculatedusing known methods for predicting melting temperatures (see, e.g.,Baldino et al., Methods Enzymology 168:761-777; Bolton et al., 1962,Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc. Natl.Acad. Sci. USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad. Sci.USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychlik etal., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, Nucleic AcidsRes 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit. Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered ketoreductase enzyme of the present disclosure.

“Hybridization stringency” relates to such washing conditions of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature Tm as determined under the solution condition for a definedpolynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Other high stringency hybridization conditions,as well as moderately stringent conditions, are described in thereferences cited above.

“Recombinant” when used with reference to, e.g., a cell, nucleic acid,or polypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature, or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques. Non-limiting examples include, amongothers, recombinant cells expressing genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise expressed at a different level.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theketoreductases enzymes may be codon optimized for optimal productionfrom the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariat analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polypeptide of thepresent disclosure. Each control sequence may be native or foreign tothe nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the expression of a polynucleotide and/or polypeptide.

“Promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of the coding region. The control sequence maycomprise an appropriate promoter sequence. The promoter sequencecontains transcriptional control sequences, which mediate the expressionof the polypeptide. The promoter may be any nucleic acid sequence whichshows transcriptional activity in the host cell of choice includingmutant, truncated, and hybrid promoters, and may be obtained from genesencoding extracellular or intracellular polypeptides either homologousor heterologous to the host cell.

“Cofactor regeneration system” refers to a set of reactants thatparticipate in a reaction that reduces the oxidized form of the cofactor(e.g., NADP+ to NADPH). Cofactors oxidized by theketoreductase-catalyzed reduction of the keto substrate are regeneratedin reduced form by the cofactor regeneration system. Cofactorregeneration systems comprise a stoichiometric reductant that is asource of reducing hydrogen equivalents and is capable of reducing theoxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme catalyst, thatcatalyzes the reduction of the oxidized form of the cofactor by thereductant. Cofactor regeneration systems to regenerate NADH or NADPHfrom NAD+ or NADP+, respectively, are known in the art and may be usedin the methods described herein.

5.2. Ketoreductase Polypeptides and Uses Thereof

The present disclosure provides engineered ketoreductase (“KRED”)polypeptides that are enzymes capable of stereospecifically reducingN-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one (“the substrate”)(e.g. compound (1) where the protecting group is a BOC moiety) to thecorresponding stereoisomeric alcohol product N-protected(2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol (“the product”) (e.g.compound (2)), as depicted in Scheme 1, above). In certain embodimentsthe substrate N-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one (“thesubstrate”) that is converted to the stereoisomeric alcohol productN-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol product (e.g.compound (2)) is present within the biocatalytic reduction reaction aspart of a racemic mixture, or as a substantially chirally pure compound,or as a chirally pure compound. The engineered ketoreductase (“KRED”)enzymes of the present disclosure are those having an improved propertywhen compared with a naturally-occurring, wild-type KRED enzyme obtainedfrom Novosphingobium aromaticivorans (SEQ ID NO:2). Enzyme propertiesfor which improvement is desirable include, but are not limited to,enzymatic activity, thermal stability, pH activity profile, cofactorrequirements, refractoriness to inhibitors (e.g., product inhibition),sterospecificity, stereoselectivity, and solvent stability. Theimprovements can relate to a single enzyme property, such as enzymaticactivity, or a combination of different enzyme properties, such asenzymatic activity and stereospecificity.

As noted above, the engineered ketoreductase with improved enzymeproperty is described with reference to Novosphingobium aromaticivorans(SEQ ID NO:2). The amino acid residue position is determined in theseketoreductases beginning from the initiating methionine (M) residue(i.e., M represents residue position 1), although it will be understoodby the skilled artisan that this initiating methionine residue may beremoved by biological processing machinery, such as in a host cell or invitro translation system, to generate a mature protein lacking theinitiating methionine residue. The amino acid residue position at whicha particular amino acid or amino acid change is present in an amino acidsequence is sometimes describe herein in terms “Xn”, or “residue n”,where n refers to the residue position. A substitution mutation, whichis a replacement of an amino acid residue in a residue corresponding toa residue of a reference sequence, for example the naturally occurringketoreductase of SEQ ID NO:2, with a different amino acid residue isdenoted as follows “X (number) Z,” where X is the amino acid found inthe wild type enzyme of N. aromaticivorans (SEQ ID NO:2) at position“number” and Z is the amino acid found at position “number” of the“mutant” enzyme, i.e. that in which amino acid Z has been substitutedfor amino acid X. In such instances, the single letter codes are used torepresent the amino acid; e.g. G145A refers to an instance in which the“wild type” amino acid glycine at position 145 of SEQ ID NO: 2 has beenreplaced with the amino acid alanine.

Herein, mutations are sometimes described as a mutation of a residue “toa” type of amino acid. For example, SEQ ID NO: 2, residue 199(isoleucine (I)) can be mutated “to a” polar residue. But the use of thephrase “to a” does not exclude mutations from one amino acid of a classto another amino acid of the same class. For example, residue 199 can bemutated from isoleucine “to an” asparagine.

A polynucleotide sequence encoding a naturally occurring ketoreductaseof Novosphingobium aromaticivorans (also referred to as “ADH” or“alcohol dehydrogenase”) can be obtained from the 780 bp region frombase 160464 to 161243 complete sequence of Novosphingobiumaromaticivorans DSM 12444 plasmid pNL2 (sequence) provided in GenBankaccession no. CP000677.1. The corresponding polypeptide sequence encodedby this polynucleotide is provided by GenBank accession no.gi|145322460|gb|ABP64403.1|[145322460]. This polypeptide is four aminoacids shorter than SEQ ID NO: 2 due to a different choice of initiationcodon (i.e., the GenBank polypeptide sequence initiates with the Metcorresponding to position 5 of SEQ ID NO: 2). The present disclosure isintended to include ketoreductase polypeptides wherein the polypeptideis a fragment of SEQ ID NO: 2, wherein the fragment amino acid sequencestarts at the Met at position 5 of SEQ ID NO: 2 and ends at position 263of SEQ ID NO: 2. Accordingly, in any of the embodiments of theengineered ketoreductase polypeptides disclosed herein, wherein thepolypeptide comprises amino acid differences relative to SEQ ID NO: 2,the disclosure also provides a fragment of an engineered ketoreductasepolypeptide, wherein the fragment amino acid sequence starts at the Metat position 5 of SEQ ID NO: 2 and ends at position 263 of SEQ ID NO: 2,and the amino acid differences are at the same amino acids as in thecorresponding full-length engineered polypeptide relative to SEQ IDNO:2.

In some embodiments, the ketoreductase polypeptides herein can have anumber of modifications relative to the reference sequence(Novosphingobium aromaticivorans of SEQ ID NO: 2) wherein themodifications result in an improved ketoreductase enzyme property. Insuch embodiments, the number of modifications to the amino acid sequencecan comprise one or more amino acids, 2 or more amino acids, 3 or moreamino acids, 4 or more amino acids, 5 or more amino acids, 6 or moreamino acids, 8 or more amino acids, 9 or more amino acids, 10 or moreamino acids, 15 or more amino acids, or 20 or more amino acids, up to10% of the total number of amino acids, up to 10% of the total number ofamino acids, up to 20% of the total number of amino acids, or up to 30%of the total number of amino acids of the reference enzyme sequence. Insome embodiments, the number of modifications to the naturally occurringpolypeptide or an engineered polypeptide that produces an improvedketoreductase property may comprise from about 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-25, 1-30, 1-35 or about 1-40 modifications of the referencesequence. The modifications can comprise insertions, deletions,substitutions, or combinations thereof.

In some embodiments, the modifications comprise amino acid substitutionsto the reference sequence, i.e., the Novosphingobium aromaticivoransKRED sequence of SEQ ID NO: 2. Substitutions that can produce animproved ketoreductase property may be at one or more amino acids, 2 ormore amino acids, 3 or more amino acids, 4 or more amino acids, 5 ormore amino acids, 6 or more amino acids, 7 or more amino acids, 8 ormore amino acids, 9 or more amino acids, 10 or more amino acids, 15 ormore amino acids, or 20 or more amino acids, up to 10% of the totalnumber of amino acids, up to 15% of the total number of amino acids, upto 20% of the total number of amino acids, or up to 30% of the totalnumber of amino acids of the reference enzyme sequence. In someembodiments, the number of substitutions to the naturally occurringpolypeptide or an engineered polypeptide that produces an improvedketoreductase property can comprise from about 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-25, 1-30, 1-35 or about 1-40 amino acid substitutions of thereference sequence.

In some embodiments, the improved property of the ketoreductasepolypeptide is with respect to an increase of its stereospecificity. Forexample, in some embodiments, the improved property is the ability ofthe enzyme to differentiate the two enantiomers of N-protected3-amino-1-chloro-4-phenylbutan-2-one (e.g., a racemic mixture of the(3S) and the (3R) enantiomers according to Formula (IV)), and convertsubstantially only the (3S) enantiomer to the correspondingstereoisomeric N-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-olproduct of Formula (II) (e.g. compound (2)), as depicted, e.g. in Scheme5, below.

This improvement in stereospecificity of the ketoreductase polypeptidecan be expressed as an improvement in the “E value” for the reactionwith the ketoreductase.

In some embodiments, the improved property of the ketoreductasepolypeptide is with respect to an increase in its ability to convert orreduce a greater percentage of the substrate to the product. In someembodiments, the improved property of the ketoreductase polypeptide iswith respect to an increase in its rate of conversion of the substrateto the product. This improvement in enzymatic activity can be manifestedby the ability to use less of the improved polypeptide as compared tothe wild-type or other reference sequence(s) to reduce or convert thesame amount of product. In some embodiments, the improved property ofthe ketoreductase polypeptide is with respect to its stability orthermostability. In some embodiments, the ketoreductase polypeptide hasmore than one improved property, such as a combination ofstereospecificity, enzyme activity, and thermostability.

In some embodiments, the ketoreductase polypeptide is capable ofstereospecifically converting the (3S)-enantiomer of N-protected3-amino-1-chloro-4-phenylbutan-2-one to give the correspondingN-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol product with apercent diastereomeric excess of at least about 25%, 50%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or99.99%.

In some embodiments, the ketoreductase polypeptide is capable ofstereospecifically converting the substrate to the product in greaterthan about 90% diastereomeric excess. Exemplary polypeptides with suchstereospecificity include, but are not limited to, the polypeptidescomprising the amino acid sequences corresponding to SEQ ID NO:4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, and 58.

In some embodiments, the ketoreductase polypeptide is capable ofstereospecifically converting the substrate to the product in greaterthan about 95% diastereomeric excess. Exemplary polypeptides with suchstereospecificity include, but are not limited to, the polypeptidescomprising the amino acid sequences corresponding to SEQ ID NO:4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, and 58.

In some embodiments, the ketoreductase polypeptide is capable ofstereospecifically converting the substrate to the product in greaterthan about 97% diastereomeric excess. Exemplary polypeptides with suchstereospecificity include, but are not limited to, the polypeptidescomprising the amino acid sequences corresponding to SEQ ID NO:4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, and 58.

In some embodiments, the ketoreductase polypeptide is capable ofstereospecifically converting the substrate to the product in greaterthan about 98% diastereomeric excess. Exemplary ketoreductasepolypeptides with such high stereospecificity include, but are notlimited to, the polypeptides comprising the amino acid sequencescorresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58.

In some embodiments, the ketoreductase polypeptide is capable ofstereospecifically converting the substrate to the product with apercent diastereomeric excess that is at least about 98%, 99%, 99.9%, or99.99%, where the polypeptide comprises an amino acid sequencecorresponding to: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58.

In some embodiments, the ketoreductase polypeptides are equivalent to orimproved as compared to wild-type (SEQ ID NO:2) with respect to theirrate of enzymatic activity, i.e., their rate or ability of convertingthe substrate to the product. Exemplary polypeptides that are capable ofconverting the substrate to the product at a conversion rate that isequivalent to or improved over wild-type, include but are not limitedto, polypeptides that comprise the amino acid sequences corresponding toSEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58.

In some embodiments, the ketoreductase polypeptides are improved ascompared to wild-type (SEQ ID NO:2) with respect to their rate ofenzymatic activity, i.e., their rate or ability of converting thesubstrate to the product. Exemplary polypeptides that are capable ofconverting the substrate to the product at a conversion rate that is atleast about 1.2-fold improved over wild-type, include but are notlimited to, polypeptides that comprise the amino acid sequencescorresponding to SEQ ID NO:4, 6, 14, 16, 18, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 50, 52, 54, and 56.

In some embodiments, the ketoreductase polypeptides are improved ascompared to wild-type (SEQ ID NO:2) with respect to their rate ofenzymatic activity, i.e., their rate or ability of converting thesubstrate to the product. Exemplary polypeptides that are capable ofconverting the substrate to the product at a conversion rate that is atleast about 1.5-fold improved over wild-type, include but are notlimited to, polypeptides that comprise the amino acid sequencescorresponding to SEQ ID NO: 6, 18, 22, 30, 38, 40, 50, 52, 54, and 56.

In some embodiments, the ketoreductase polypeptides are improved ascompared to wild-type (SEQ ID NO:2) with respect to their rate ofenzymatic activity, i.e., their rate or ability of converting thesubstrate to the product. Exemplary polypeptides that are capable ofconverting the substrate to the product at a conversion rate that is atleast about 3-fold improved over wild-type, include but are not limitedto, polypeptides that comprise the amino acid sequences corresponding toSEQ ID NO:6, 50, 52, and 56.

In some embodiments, the engineered ketoreductase polypeptides of thedisclosure are capable of converting the substrate to product with adiastereomeric excess of at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.9%, 99.99%, or more. Exemplary engineeredketoreductase polypeptides that have this capability include, but arenot limited to, polypeptides comprising the sequence that corresponds toSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 and 58.

In some embodiments, the engineered ketoreductase polypeptides of thedisclosure are capable of improved conversion rates for reducing asubstrate compound of Formula (I) (e.g., compound (1)) to a productcompound of Formula (II) (e.g., compound (2)). For example, in someembodiments, the engineered ketoreductase polypeptides of the disclosureare capable of converting at least about 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of thesubstrate to the product in about 24 hours or less. In some embodiments,the engineered ketoreductase polypeptides are capable of converting atleast about 90% of the substrate to the product in less than about 24hours, less than about 20 hours, less than about 16 hours, less thanabout 12 hours, and even less than about 10 hours. Exemplary engineeredketoreductase polypeptides that have this capability include, but arenot limited to, polypeptides comprising the sequence that corresponds toSEQ ID NO: 6, 50, 52, and 56.

In some embodiments, the engineered ketoreductase polypeptides of thedisclosure are capable of converting at least about 70%, 80%, 90%, 95%,99% or more of a substrate of compound of Formula (I) (e.g., compound(1)) to a product compound of Formula (II) (e.g., compound (2)) in about24 hours or less when the reaction mixture comprises about 1% or less(but more than 0%), 0.5% or less (but more than 0%), 0.2% or less (butmore than 0%), or even 0.1% or less (but more than 0%) of theketoreductase polypeptide by weight with respect to the weight of theamount of substrate. Exemplary polypeptides that have this capabilityinclude, but are not limited to, polypeptides comprising the sequencethat corresponds to SEQ ID NO: 6, 50, 52, and 56.

In some embodiments, the engineered ketoreductase polypeptides of thedisclosure are capable of converting at least about 70%, 80%, 90%, 95%,99% or more of a substrate of compound of Formula (I) (e.g., compound(1)) to a product compound of Formula (II) (e.g., compound (2)) in about24 hours or less when the reaction mixture comprises a ketoreductasepolypeptide loading of about 10 g/L or less, 5 g/L or less, 2 g/L orless, 1 g/L or less and an initial concentration of substrate in thereaction mixture (i.e., substrate loading) of at least about 25 g/L, atleast about 50 g/L, at least about 75 g/L, at least about 100 g/L, atleast about 125 g/L, at least about 150 g/L, at least about 175 g/L, orat least about 200 g/L. Exemplary polypeptides that have this capabilityinclude, but are not limited to, polypeptides comprising the sequencethat corresponds to SEQ ID NO: 6, 50, 52, and 56.

In some embodiments, the ketoreductase polypeptides have improvedactivity and stability over wild-type, and can reduce the substrate tothe product in greater than about 98% d.e. Exemplary polypeptides withsuch capabilities include, but are not limited to SEQ ID NO: 4, 6, 14,16, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 50, 52, 54, and 56.Table 2 below lists engineered ketoreductase polypeptides (and encodingpolynucleotides) by sequence identifier (SEQ ID NO) disclosed hereintogether with the specific residue differences of the variant sequencesof the engineered polypeptides relative to the wild-type Novosphingobiumaromaticivorans ketoreductase sequences (SEQ ID NO:2) from which theywere derived by directed evolution (see e.g., Stemmer et al., 1994, ProcNatl Acad Sci USA 91:10747-10751). Each row of Table 2 lists two SEQ IDNOs, where the odd number refers to the nucleotide sequence that encodesfor the polypeptide amino acid sequence provided by the even number.

The activity of each engineered ketoreductase polypeptide was determinedrelative to the activity wild-type enzyme of SEQ ID NO: 2 (wild-type:˜30% conversion in 24 hours, 3 g/L substrate loading, 5 g/L enzymeloading). Activity was determined as conversion of compound (1) tocompound (2) monitored over time, as described in Example 7. Assummarized in Table 2, the improvement in activity was quantified asfollows: “Control” indicates 100% to 120% as compared to the activity ofKRED of SEQ ID NO:2; “+” indicates >120% to 150% as compared to the KREDof SEQ ID NO:2; “++” indicates >150% to 300% as compared to the KRED ofSEQ ID NO:2; and “+++” indicates >300% as compared to the KRED of SEQ IDNO:2.

TABLE 2 SEQ ID Improvement NO: Residue Difference(s) in (nt/aa)(relative to SEQ ID NO: 2) Activity 3/4 G145A; + 5/6 G145A; I199L; +++7/8 S90V; A232V Control  9/10 F152L Control 11/12 A34S Control 13/14A47V; I199M + 15/16 I199L + 17/18 N153G ++ 19/20 N153V Control 21/22N153H ++ 23/24 A150G + 25/26 I91L + 27/28 I91W + 29/30 I91R ++ 31/32I91K + 33/34 V144T + 35/36 A150G; P231F + 37/38 G190A ++ 39/40 F260Y ++41/42 S198N Control 43/44 D81N; M200I Control 45/46 G145A; S261N Control47/48 V28A; D112Y; G117D; S143R; G145A; R148H; Control E233Q; S261N49/50 P2L; E50K; G145A; A217T +++ 51/52 G145A +++ 53/54 K94R; G145A;I199N ++ 55/56 G145A; I225V +++ 57/58 G145A; T158S; D244G Control

The improved activity of the engineered ketoreductase polypeptides forthe conversion of the secondary alcohol, isopropanol (IPA) to itscorresponding product, acetone was determined relative to the sameactivity for the reference polypeptide of SEQ ID NO: 2. Relative IPAactivity was determined using an assay with the following reactionconditions: 100 μl 10× diluted engineered KRED lysate, 10% IPA (v/v),0.5 g/L NAD⁺, 100 mM TEA, pH 7.5. Exemplary engineered ketoreductasepolypeptides exhibiting at least 2-fold increased activity with IPArelative to SEQ ID NO: 2 are listed in Table 3. The fold-improvement inIPA activity relative the WT of SEQ ID NO: 2 was quantified as follows:“+” indicates at least 200% to 250% improvement; “++” indicates <250% to500% improvement; and “+++” indicates >500% to 1000% improvement; and“++++” indicates >1000% to 2000% improvement.

TABLE 3 SEQ ID Residue Differences FIOP in IPA NO: (relative to SEQ IDNO: 2) activity  6 G145A, I199L ++ 56 G145A, I225V ++ 60 G145V + 62I199G ++ 64 V144C ++ 66 A150S ++ 68 A150I ++ 70 A150W +++ 72 G190P ++ 74G190Q + 76 G190V ++++ 78 V204F ++ 80 M200I +++

In some embodiments, the present disclosure provides an improvedketoreductase polypeptide comprising an amino acid sequence that is atleast about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO:2 and comprising at leastone amino acid substitution listed in Table 2 or Table 3.

In some embodiments, the present disclosure provides an improvedketoreductase polypeptide capable of exhibiting a relative activity atleast about 3-fold that of a polypeptide of SEQ ID NO: 2 (i.e., “+++”based on relative activity designations of Table 2 above), wherein theimproved ketoreductase polypeptide comprises an amino acid sequence thatis at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 and comprises atleast one of the following amino acid substitutions or sets of aminoacid substitutions: G145A; G145A and 1225V; P2L, E50K, G145A, and A217T;G145A, and I199L. Such improved ketoreductase polypeptides disclosedherein may further comprise additional modifications, includingsubstitutions, deletions, insertions, or combinations thereof. Thesubstitutions can be non-conservative substitutions, conservativesubstitutions, or a combination of non-conservative and conservativesubstitutions. Other useful amino acid sequence substitutions forimproved ketoreductases at positions P2, E50, G145, I199, A217, and1225, are disclosed below. In some embodiments, these ketoreductasepolypeptides can have optionally from about 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-25, 1-30, 1-35 or about 1-40 mutations at other amino acidresidues. In some embodiments, the number of modifications can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35or about 40 other amino acid residues.

In some embodiments, the present disclosure provides an improvedketoreductase polypeptide capable of exhibiting a relative activity atleast about 1.5-fold that of a polypeptide of SEQ ID NO: 2 (i.e., “++”based on relative activity designations of Table 2 above), wherein theimproved ketoreductase polypeptide comprises an amino acid sequence thatis at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 and comprises atleast one of the following amino acid substitutions as compared to SEQID NO:2: N153G; N153H; G190A; F260Y; I91R; K94R; G145A; I199N; or thefollowing set of amino acid substitutions as compared to SEQ ID NO:2:K94R, G145A, and I199N. Such improved ketoreductase polypeptidesdisclosed herein may further comprise additional modifications,including substitutions, deletions, insertions, or combinations thereof.The substitutions can be non-conservative substitutions, conservativesubstitutions, or a combination of non-conservative and conservativesubstitutions. Other useful amino acid sequence substitutions forimproved ketoreductases at positions I91, K94, G145, N153, G190, I199,and F260, are disclosed below. In some embodiments, these ketoreductasepolypeptides can have optionally from about 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-25, 1-30, 1-35 or about 1-40 mutations at other amino acidresidues. In some embodiments, the number of modifications can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35or about 40 other amino acid residues.

In some embodiments, the present disclosure provides an improvedketoreductase polypeptide capable of exhibiting a relative activity atleast about 1.2-fold that of a polypeptide of SEQ ID NO: 2 (i.e., “+”based on relative activity designations of Table 2 above), wherein theimproved ketoreductase polypeptide comprises an amino acid sequence thatis at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 and comprises atleast one of the following amino acid substitutions as compared to SEQID NO:2: I199L; A150G; I91L; I91W; I91K; and V144T; or one of thefollowing sets of amino acid substitutions as compared to SEQ ID NO:2:G145A; A47V and I199M; A150G and P231F. Such improved ketoreductasepolypeptides disclosed herein may further comprise additionalmodifications, including substitutions, deletions, insertions, orcombinations thereof. The substitutions can be non-conservativesubstitutions, conservative substitutions, or a combination ofnon-conservative and conservative substitutions. Other useful amino acidsequence substitutions for improved ketoreductases at positions A47,I91, V144, G145, A150, I199, and P231, are disclosed below. In someembodiments, these ketoreductase polypeptides can have optionally fromabout 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40mutations at other amino acid residues. In some embodiments, the numberof modifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15,16, 18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.

In some embodiments, the present disclosure provides an improvedketoreductase polypeptide capable of exhibiting a relative activity inconverting isopropanol to acetone of at least about 2-fold that of apolypeptide of SEQ ID NO: 2 (i.e., “+” based on relative activitydesignations of Table 3 above), wherein the improved ketoreductasepolypeptide comprises an amino acid sequence that is at least about 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO:2 and comprises at least one of the followingamino acid substitutions as compared to SEQ ID NO:2: V144C, G145A,G145V, A1505, A1501, A150W, G190P, G190Q, G190V, I199G, I199L, M200I,V204F, or 1225V; or one of the following sets of amino acidsubstitutions as compared to SEQ ID NO:2: G145A, I199L; or G145A, 1225V.Such improved ketoreductase polypeptides disclosed herein may furthercomprise additional modifications, including substitutions, deletions,insertions, or combinations thereof. The substitutions can benon-conservative substitutions, conservative substitutions, or acombination of non-conservative and conservative substitutions. Otheruseful amino acid sequence substitutions for improved ketoreductases atpositions V144, G145, A150, G190, I199, M200, V204, and 1225, aredisclosed below. In some embodiments, these ketoreductase polypeptidescan have optionally from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9,1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30,1-35 or about 1-40 mutations at other amino acid residues. In someembodiments, the number of modifications can be 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 otheramino acid residues.

Accordingly, in some embodiments the present disclosure provides anengineered ketoreductase polypeptide capable of oxidizing isopropanol(IPA) to acetone with an activity at least 2-fold, 2.5-fold, 5-fold, or10-fold greater than the reference polypeptide of SEQ ID NO: 2, whereinthe polypeptide comprises an amino acid sequence having at least 70%,80%, 85%, 90%, 95%, 98%, 99% or greater identity to a sequence selectedfrom SEQ ID NO: 6, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 80.In some embodiments, the engineered polypeptide has an amino acidsequence that has at least 95% identity to a sequence selected from SEQID NO: 6, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 80. In someembodiments, the engineered polypeptide has an amino acid sequenceselected from SEQ ID NO: 6, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,and 80.

In some embodiments, the engineered ketoreductase polypeptide capable ofoxidizing isopropanol (IPA) to acetone with an activity at least 2-fold,2.5-fold, 5-fold, or 10-fold greater than the reference polypeptide ofSEQ ID NO: 2, has an amino acid sequence comprises at least one of thefollowing features: residue corresponding to amino acid 144 of SEQ IDNO:2 is cysteine; residue corresponding to amino acid 145 of SEQ ID NO:2is selected from the group consisting of alanine, and valine; residuecorresponding to amino acid 150 of SEQ ID NO:2 is selected from thegroup consisting of isoleucine, serine, and tryptophan; residuecorresponding to amino acid 190 of SEQ ID NO:2 is selected from thegroup consisting of glutamine, proline, and valine; residuecorresponding to amino acid 199 of SEQ ID NO:2 is selected from thegroup consisting of glycine, and leucine; residue corresponding to aminoacid 200 of SEQ ID NO:2 is isoleucine; residue corresponding to aminoacid 204 of SEQ ID NO:2 is phenylalanine; and residue corresponding toamino acid 225 of SEQ ID NO:2 is valine. In certain embodiments, theamino acid sequence of the engineered polypeptide comprises at least oneof the following substitutions as compared to SEQ ID NO:2: V144C, A150I,A150S, A150W, G190P, G190V, M200I, and V204F. In certain embodiments,the amino acid sequence of the engineered polypeptide comprises at leastone of the following sets of amino acid substitutions as compared to SEQID NO:2: G145A, and I199L; and G145A, and 1225V.

In some embodiments, the engineered ketoreductase polypeptide is capableof oxidizing isopropanol (IPA) to acetone with an activity at least5-fold greater than the reference polypeptide of SEQ ID NO: 2, andwherein the amino acid sequence comprises at least one of the followingsubstitutions as compared to SEQ ID NO:2: A150W, M200I, and G190V.

In some embodiments, the ketoreductase polypeptides of the presentdisclosure can have one or more modifications (i.e., residuedifferences) as compared to the reference amino acid sequence or ascompared to any of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, or 80. The modifications can includesubstitutions, deletions, insertions, or combinations thereof. Thesubstitutions can be non-conservative substitutions, conservativesubstitutions, or a combination of non-conservative and conservativesubstitutions. In some embodiments, these ketoreductase polypeptides canhave optionally from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35or about 1-40 mutations at other amino acid residues. In someembodiments, the number of modifications can be 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35 or about 40 otheramino acid residues.

In some embodiments the present disclosure provides an improvedketoreductase polypeptide comprising an amino acid sequence that is atleast about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO:2, and comprise at leastone amino acid substitution as compared to SEQ ID NO: 2 selected fromthe group consisting of: the proline residue at position 2 is replacedwith an aliphatic or nonpolar amino acid selected from among alanine,leucine, valine, isoleucine, glycine and methionine; the valine residueat position 28, in a conservative change, is replaced with an aliphaticor nonpolar amino acid selected from among alanine, leucine, valine,isoleucine, glycine and methionine; the alanine residue at position 34is replaced with a polar amino acid selected from among asparagine,glutamine, serine, and threonine; the alanine residue at position 47, ina conservative change, is replaced with an aliphatic or nonpolar aminoacid selected from among alanine, leucine, valine, isoleucine, glycineand methionine; the glutamic acid residue at position 50 is replacedwith a basic amino acid selected from lysine and arginine; the asparticacid residue at position 81 is replaced with a polar amino acid selectedfrom among asparagine, glutamine, serine, and threonine; the serineresidue at position 90 is replaced with an aliphatic or nonpolar aminoacid selected from among alanine, leucine, valine, isoleucine, glycineand methionine; the isoleucine residue at position 91 is, in aconservative change, replaced with an aliphatic or nonpolar amino acidselected from among alanine, leucine, valine, isoleucine, glycine andmethionine, while in other aspects, the isoleucine residue at position91 is replaced with an aromatic amino acid selected from among tyrosine,tryptophan, and phenylalanine, or a basic amino acid selected from amonglysine and arginine; the lysine residue at position 94 is, in aconservative change, replaced with another basic amino acid, arginine;the aspartic acid residue at position 112 is replaced with an aromaticamino acid selected from among tyrosine, tryptophan, and phenylalanine;the glycine residue at position 117 is replaced with an acidic aminoacid selected from among aspartic acid and glutamic acid; the serineresidue at position 143 is replaced with a basic amino acid selectedfrom among lysine and arginine; the valine residue at position 144 isreplaced with a cysteine or a polar amino acid selected from amongasparagine, glutamine, serine, and threonine; the glycine residue atposition 145, in either a conservative or non-conservative change, maybe replaced with a nonpolar amino acid selected from among alanine,leucine, valine, isoleucine, and methionine or an aliphatic amino acidselected from among alanine, leucine, valine, isoleucine; the arginineresidue at position 148 is replaced with a constrained amino acidselected from among proline and histidine; the alanine residue atposition 150, in conservative or non-conservative change, is replacedwith another nonpolar or aliphatic amino acid selected from amongleucine, valine, isoleucine, glycine and methionine, or a polar aminoacid selected from among asparagine, glutamine, serine, and threonine,or an aromatic amino acid selected from among tyrosine, tryptophan, andphenylalanine; the phenylalanine residue at position 152 is replacedwith a nonpolar or aliphatic amino acid selected from among alanine,leucine, valine, isoleucine, glycine and methionine; the asparagineresidue at position 153 is replaced with a nonpolar or aliphatic aminoacid selected from among alanine, leucine, valine, isoleucine, glycineand methionine, or with a constrained amino acid selected from amonghistidine and proline; the threonine residue at position 158, in aconservative change, is replaced with another polar amino acid selectedfrom among asparagine, glutamine, and serine; the glycine residue atposition 190, in either a conservative or a nonconservative change, isreplaced with either a nonpolar or an aliphatic amino acid selected fromamong alanine, valine, leucine, isoleucine, glycine, and methionine, ora polar amino acid selected from among asparagine, glutamine, andserine, or a proline; the serine residue at position 198, in aconservative change, is replaced with another polar amino acid selectedfrom among asparagine, glutamine, and threonine; the isoleucine residueat position 199 is, in a conservative change replaced with anotheraliphatic or nonpolar amino acid selected from among alanine, leucine,valine, glycine, and methionine, or with a polar amino acid selectedfrom among asparagine, glutamine, serine, and threonine; the methionineresidue at position 200, in a conservative change, is replaced withanother nonpolar amino acid selected from among alanine, leucine,valine, isoleucine, and glycine; the valine at position 204, in anon-conservative change, is replaced with an aromatic amino acidselected from among tyrosine, tryptophan, and phenylalanine; the alanineresidue at position 217 is replaced with a polar amino acid selectedfrom among asparagine, glutamine, serine and threonine; the isoleucineresidue at position 225, in a conservative change, is replaced withanother nonpolar amino acid selected from among valine, leucine,glycine, and methionine; the proline residue at position 231 is replacedwith an aromatic amino acid selected from among tyrosine, tryptophan,and phenylalanine; the alanine residue at position 232, in aconservative change, is replaced with another nonpolar amino acidselected from among leucine, isoleucine, valine, glycine, andmethionine; the glutamic acid residue at position 233 is replaced with apolar amino acid selected from among asparagine, glutamine, serine, andthreonine; the aspartic acid residue at position 244 is replaced with anonpolar amino acid selected from among alanine, leucine, isoleucine,valine, glycine, and methionine; the phenylalanine residue at position260, in a conservative change, is replaced with another aromatic aminoacid selected from among tyrosine and tryptophan; and the serine residueat position 261, in a conservative change, is replaced with anotherpolar amino acid selected from among asparagine, glutamine, andthreonine. The forgoing improved ketoreductase polypeptides may furthercomprise additional modifications, including substitutions, deletions,insertions, or combinations thereof. The substitutions can benon-conservative substitutions, conservative substitutions, or acombination of non-conservative and conservative substitutions. In someembodiments, these ketoreductase polypeptides can have optionally fromabout 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40mutations at other amino acid residues. In some embodiments, the numberof modifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15,16, 18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.

In certain embodiments, the improved ketoreductase polypeptides of thepresent disclosure comprise an amino acid sequence that is at leastabout 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO:2, and comprise, as compared toSEQ ID NO:2, at least one amino acid substitution selected from thegroup consisting of: the proline residue at position 2 is replaced withleucine (P2L); the valine residue at position 28 is replaced withalanine (V28A); the alanine residue at position 34 is replaced withserine (A34S); the alanine residue at position 47 is replaced withvaline (A47V); the glutamic acid residue at position 50 is replaced withlysine (E50K); the aspartic acid residue at position 81 is replaced withasparagine (D81N); the serine residue at position 90 is replaced withvaline (590V); the isoleucine residue at position 91 is replaced with anamino acid selected from among leucine (I91L), tryptophan (I91W),arginine (I91R), and lysine (I91K); the lysine residue at position 94 isreplaced with arginine (K94R); the aspartic acid residue at position 112is replaced with tyrosine (D112Y); the glycine residue at position 117is replaced with aspartic acid (G117D); the serine residue at position143 is replaced with arginine (S143R); the valine residue at position144 is replaced with an amino acid selected from among cysteine (V144C)and threonine (V144T); the glycine residue at position 145 is replacedwith an amino acid selected from among alanine (G145A) and valine(G145V); the arginine residue at position 148 is replaced with histidine(R148H); the alanine residue at position 150 is replaced with an aminoacid selected from among glycine (A150G), isoleucine (A150I), serine(A150S), and tryptophan (A150W); the phenylalanine residue at position152 is replaced with leucine (F152L); the asparagine residue at position153 is replaced with an amino acid selected from among glycine (N153G),valine (N153V), and histidine (N153H); the threonine residue at position158 is replaced with serine (T158S); the glycine residue at position 190is replaced with an amino acid selected from among alanine (G190A),proline (G190P), glutamine (G190Q), and valine (G190V); the serineresidue at position 198 is replaced with asparagine (S198N); theisoleucine residue at position 199 is replaced with an amino acidselected from among glycine (I199G), methionine (I199M), leucine(I199L), and asparagine (I199N); the methionine residue at position 200is replaced with isoleucine (M200I); the valine residue at position 204is replaced with phenylalanine (V204F); the alanine residue at position217 is replaced with threonine (A217T); the isoleucine residue atposition 225 is replaced with valine (I225V); the proline residue atposition 231 is replaced with phenylalanine (P231F); the alanine residueat position 232 is replaced with valine (A232V); the glutamic acidresidue at position 233 is replaced with glutamine (E233Q); the asparticacid residue at position 244 is replaced with glycine (D244G); thephenylalanine residue at position 260 is replaced with tyrosine (F260Y);and the serine residue at position 261 is replaced with asparagine(S261N).

In certain embodiments, an engineered ketoreductase polypeptide of thepresent disclosure comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, and 80. The foregoing improvedketoreductase polypeptides may further comprise additionalmodifications, including substitutions, deletions, insertions, orcombinations thereof. The substitutions can be non-conservativesubstitutions, conservative substitutions, or a combination ofnon-conservative and conservative substitutions. In some embodiments,these ketoreductase polypeptides can have optionally from about 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40 mutations atother amino acid residues. In some embodiments, the number ofmodifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.

In some embodiments, the improved engineered ketoreductase enzymes cancomprise deletions of the naturally occurring ketoreductase polypeptidesas well as deletions of other improved ketoreductase polypeptides. Insome embodiments, each of the improved engineered ketoreductase enzymesdescribed herein can comprise deletions of the polypeptides describedherein. Thus, for each and every embodiment of the ketoreductasepolypeptides of the disclosure, the deletions can comprise one or moreamino acids, 2 or more amino acids, 3 or more amino acids, 4 or moreamino acids, 5 or more amino acids, 6 or more amino acids, 8 or moreamino acids, 10 or more amino acids, 15 or more amino acids, or 20 ormore amino acids, up to 10% of the total number of amino acids, up to10% of the total number of amino acids, up to 20% of the total number ofamino acids, or up to 30% of the total number of amino acids of theketoreductase polypeptides, as long as the functional activity of theketoreductase activity is maintained. In some embodiments, the deletionscan comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40amino acid residues.

As described herein, the ketoreductase polypeptides of the disclosurecan be in the form of fusion polypeptides in which the ketoreductasespolypeptides are fused to other polypeptides, such as antibody tags(e.g., myc epitope) or purifications sequences (e.g., His tags). Thus,the ketoreductase polypeptides can be used with or without fusions toother polypeptides.

In some embodiments, the improved engineered ketoreductase enzymes cancomprise additions or insertions of amino acid sequences the naturallyoccurring ketoreductase polypeptides as well as additions to orinsertions of amino acid sequences to other improved ketoreductasepolypeptides. In particular embodiments, a ketoreductases polypeptide ofthe present disclosure may, for example, comprise 1-20, 2-15, 3-10, 4-8,or 5-7 additional amino acid at the amino- or carboxy-terminus of thenaturally occurring ketoreductase polypeptides, as well as to improvedketoreductase polypeptides of the disclosure. For each and everyembodiment of the ketoreductase polypeptides of the disclosure, theinsertions or additions can comprise one or more amino acids, 2 or moreamino acids, 3 or more amino acids, 4 or more amino acids, 5 or moreamino acids, 6 or more amino acids, 8 or more amino acids, 10 or moreamino acids, 15 or more amino acids, or 20 or more amino acids, up to10% of the total number of amino acids, up to 10% of the total number ofamino acids, up to 20% of the total number of amino acids, or up to 30%of the total number of amino acids of the ketoreductase polypeptides, aslong as the functional activity of the ketoreductase activity ismaintained. In some embodiments, the insertions or additions cancomprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40amino acid residues.

The polypeptides described herein are not restricted to the geneticallyencoded amino acids. In addition to the genetically encoded amino acids,the polypeptides described herein may be comprised, either in whole orin part, of naturally-occurring and/or synthetic non-encoded aminoacids. Certain commonly encountered non-encoded amino acids of which thepolypeptides described herein may be comprised include, but are notlimited to: the D-enantiomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (NaI); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4 chlorophenylalanine (Pcf); 2fluorophenylalanine (Off); 3 fluorophenylalanine (Mff); 4fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N acetyl lysine (AcLys); 2,4 diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro).

Additional non-encoded amino acids of which the polypeptides describedherein may be comprised will be apparent to those of skill in the art(see, e.g., the various amino acids provided in Fasman, 1989, CRCPractical Handbook of Biochemistry and Molecular Biology, CRC Press,Boca Raton, Fla., at pp. 3-70 and the references cited therein, all ofwhich are incorporated by reference). These amino acids may be in eitherthe L or D configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N methyl amino acids (L configuration); 1 aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1 aminocyclopentane-3-carboxylic acid.

As described above the various modifications introduced into thenaturally occurring polypeptide to generate an engineered ketoreductaseenzyme can be targeted to a specific property of the enzyme.

5.3. Polynucleotides Encoding Engineered Ketoreductases

In another aspect, the present disclosure provides polynucleotidesencoding the engineered ketoreductase enzymes. The polynucleotides maybe operatively linked to one or more heterologous regulatory sequencesthat control gene expression to create a recombinant polynucleotidecapable of expressing the polypeptide. Expression constructs containinga heterologous polynucleotide encoding the engineered ketoreductase canbe introduced into appropriate host cells to express the correspondingketoreductase polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improved ketoreductaseenzymes disclosed herein. Thus, having identified a particular aminoacid sequence, those skilled in the art could make any number ofdifferent nucleic acids by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of theprotein. In this regard, the present disclosure specificallycontemplates each and every possible variation of polynucleotides thatcould be made by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide disclosed herein, including the amino acidsequences presented in Table 2.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a ketoreductase polypeptide with an amino acid sequence thathas at least about 80% or more sequence identity, at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity, or more sequence identity to any of the engineeredketoreductase polypeptides described herein, i.e., a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, and 80.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. For example, the polynucleotide of SEQ ID NO:1 could be codonoptimized for expression in E. coli, but otherwise encode the naturallyoccurring ketoreductase of Novosphingobium aromaticivorans.

In some embodiments, all codons need not be replaced to optimize thecodon usage of the ketoreductases since the natural sequence willcomprise preferred codons and because use of preferred codons may not berequired for all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the ketoreductase enzymes may contain preferredcodons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codonpositions of the full length coding region.

In some embodiments, the polynucleotides encoding the engineeredketoreductases are selected from SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79. In someembodiments, the polynucleotides encoding the engineered ketoreductasesare capable of hybridizing under highly stringent conditions to apolynucleotide comprising SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79. These polynucleotidesencode some of the polypeptides represented by the amino acid sequenceslisted in Tables 2 and 3.

In other embodiments, the polynucleotides comprise polynucleotides thatencode the polypeptides described herein but have about 80% or moresequence identity, about 85% or more sequence identity, about 90% ormore sequence identity, about 95% or more sequence identity, about 98%or more sequence identity, or 99% or more sequence identity at thenucleotide level to a reference polynucleotide encoding an engineeredketoreductase. In some embodiments, the reference polynucleotide isselected from polynucleotide sequences represented by SEQ ID NO: 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,and 79.

An isolated polynucleotide encoding an improved ketoreductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus lichenifonnis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978, Proc.Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoeret al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25).

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′-end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′-endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NC1B11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the KRED polypeptide ofthe present invention would be operably linked with the regulatorysequence.

Thus, in another embodiment, the present disclosure is also directed toa recombinant expression vector comprising a polynucleotide encoding anengineered ketoreductase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The various nucleic acid and controlsequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), cysC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Embodiments for use in an Aspergillus cell includethe amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzaeand the bar gene of Streptomyces hygroscopicus.

The expression vectors of the present invention preferably contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori) or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAMβ1 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes it's functioningtemperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, ProcNatl Acad. Sci. USA 75:1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors includep3xFLAGTMTM expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptII SK(−) and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

5.4. Host Cells for Expression of Ketoreductase Polypeptides

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved ketoreductasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe ketoreductase enzyme in the host cell. Host cells for use inexpressing the KRED polypeptides encoded by the expression vectors ofthe present invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Lactobacillus,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums and growthconditions for the above-described host cells are well known in the art.Accordingly, in some embodiments, the engineered ketoreductasepolypeptides disclosed herein can be prepared by standard methodscomprising culturing a host cell containing a suitable expression vectorcomprising the polynucleotide encoding the polypeptide.

Polynucleotides for expression of the ketoreductase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

An exemplary host cell is Escherichia coli W3110. Another exemplary hostcell is Escherichia coli BL21. The expression vector is created byoperatively linking a polynucleotide encoding an improved ketoreductaseinto the plasmid pCK110900 (see, US application publication 20040137585)operatively linked to the lac promoter under control of the ladrepressor. The expression vector also contains the P15a origin ofreplication and the chloramphenicol resistance gene. Cells containingthe subject polynucleotide in Escherichia coli W3110 or BL21 areisolated by subjecting the cells to chloramphenicol selection.

5.5. Methods of Generating Engineered Ketoreductase Polypeptides

In some embodiments, to make the improved KRED polynucleotides andpolypeptides of the present disclosure, the naturally-occurringketoreductase enzyme that catalyzes the reduction reaction is obtained(or derived) from Novosphingobium aromaticivorans. In some embodiments,the parent polynucleotide sequence is codon optimized to enhanceexpression of the ketoreductase in a specified host cell. As anillustration, the parental polynucleotide sequence encoding thewild-type KRED polypeptide of Novosphingobium aromaticivorans (SEQ IDNO:1), can be assembled from oligonucleotides based upon that sequenceor from oligonucleotides comprising a codon-optimized coding sequencefor expression in a specified host cell, e.g., an E. coli host cell. Inone embodiment, the polynucleotide can be cloned into an expressionvector, placing the expression of the ketoreductase gene under thecontrol of the lac promoter and lad repressor gene. Clones expressingthe active ketoreductase in E. coli can be identified and the genessequenced to confirm their identity.

The engineered ketoreductases can be obtained by subjecting thepolynucleotide encoding the naturally occurring ketoreductase tomutagenesis and/or directed evolution methods, as discussed above. Anexemplary directed evolution technique is mutagenesis and/or DNAshuffling as described in Stemmer, 1994, Proc Natl Acad Sci USA91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746. Other directedevolution procedures that can be used include, among others, staggeredextension process (StEP), in vitro recombination (Zhao et al., 1998,Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al., 1994, PCRMethods Appl. 3:S136-S140), and cassette mutagenesis (Black et al.,1996, Proc Natl Acad Sci USA 93:3525-3529).

The clones obtained following mutagenesis treatment are screened forengineered ketoreductases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry technique of monitoring the rate ofdecrease (via a decrease in absorbance or fluorescence) of NADH or NADPHconcentration, as it is converted into NAD+ or NADP+. In this reaction,the NADH or NADPH is consumed (oxidized) by the ketoreductase as theketoreductase reduces a ketone substrate to the corresponding hydroxylgroup. The rate of decrease of NADH or NADPH concentration, as measuredby the decrease in absorbance or fluorescence, per unit time indicatesthe relative (enzymatic) activity of the KRED polypeptide in a fixedamount of the lysate (or a lyophilized powder made therefrom). Where theimproved enzyme property desired is thermal stability, enzyme activitymay be measured after subjecting the enzyme preparations to a definedtemperature and measuring the amount of enzyme activity remaining afterheat treatments. Clones containing a polynucleotide encoding aketoreductase are then isolated, sequenced to identify the nucleotidesequence changes (if any), and used to express the enzyme in a hostcell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLeft 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, such as The Midland Certified Reagent Company, Midland, Tex.,The Great American Gene Company, Ramona, Calif., ExpressGen Inc.Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and manyothers.

Engineered ketoreductase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic BTM fromSigma-Aldrich of St. Louis Mo. Accordingly, in some embodiments, theengineered ketoreductase polypeptides disclosed herein can be preparedby standard methods comprising culturing a host cell containing asuitable expression vector comprising the polynucleotide encoding thepolypeptide and isolating the polypeptide from the host cell.

Chromatographic techniques for isolation of the ketoreductasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved ketoreductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the ketoreductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with a polypeptide of the disclosure. Thepolypeptide may be attached to a suitable carrier, such as BSA, by meansof a side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette Guerin) and Corynebacterium parvum.

5.6. Methods of Using the Engineered Ketoreductase Enzymes and CompoundsPrepared Therewith

The ketoreductase enzymes described herein can catalyze theenantiospecific reduction of the N-protected(S)-3-amino-1-chloro-4-phenylbutan-2-one compound of Formula (I) (“thesubstrate”) (e.g. compound (1) where the protecting group is a BOCmoiety) to the corresponding stereoisomeric alcohol product N-protected(2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol compound of Formula (II)(“the product”) (e.g. compound (2)), as depicted in Scheme 1 (seeabove).

In some embodiments, the invention provides a method forstereospecifically enriching an N-protected(R)-3-amino-1-chloro-4-phenylbutan-2-one compound in a mixture with anN-protected (S)-3-amino-1-chloro-4-phenylbutan-2-one by reducing thelatter ketone compound in the mixture by contacting or incubating with aketoreductase polypeptide disclosed herein under suitable reactionconditions for producing a chiral alcohol product, N-protected(2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol, as illustrated in thereaction of Scheme 5 (see above). Accordingly, in some embodiments, theketoreductase polypeptides having improved stereospecificity (comparedto SEQ ID NO: 2) of the present disclosure can be use to resolvemixtures of chiral alpha-chloroketone compounds.

In some embodiments of the method, at least about 45% of a racemicsubstrate mixture is reduced to the product in less than 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 hours when themethod is conducted with greater than or equal to 200 g/L substrate andless than or equal to 2 g/L ketoreductase enzyme (but more than 0 g/Lenzyme).

The processes for converting a compound of Formula (I) to a chiralchloroalcohol compound of Formula (II) using ketoreductase enzymesdisclosed herein represent substantial improvements over known methodsdue in part to the resulting high yield (e.g., conversion rate>80% ormore in less than 24 hours), high purity (e.g., >99% d.e.), andfavorable solvent systems that allow for a “telescoped” reaction forpreparing the compound of Formula (II) and using it as a reagent forsubsequent reaction.

In some embodiments of the methods, the product has greater than about90%, 95%, 97%, 98%, 99%, or even greater diastereomeric excess of theN-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol.

In some embodiments of the methods, about 95% of the substrate isconverted to the product in less than about 24 hours when carried outwith greater than about 100 g/L of substrate and less than about 5 g/Lof the polypeptide.

In some embodiments of the methods at least about 90%, 95%, 97%, 98%, ormore, of the compound of Formula (I) is converted to the compound ofFormula (II) in less than about 24 hours, 20 hours, 12 hours, 8 hours,or even less.

In some embodiments of the methods at least about 95% of the compound ofFormula (I) is converted to the compound of Formula (II) in less thanabout 24 hours, wherein the compound of Formula (I) concentration is atleast about 150 g/L and the polypeptide concentration is less than about1 g/L.

In certain embodiments, the present disclosure further provides a methodfor preparing an epoxide compound of Formula (III) by converting acompound of Formula (I) to a chiral chloroalcohol compound of Formula(II) (e.g., compound (2)) using a ketoreductase of the presentdisclosure, and then cyclizing the compound of Formula (II) to anepoxide compound of Formula (III) (e.g., compound (3)), according toSchemes 3 and 4, depicted above. This conversion can be carried out veryefficiently by extracting the crude enzymatic reaction mixturecontaining the compound of Formula (II) with a suitable solvent (e.g.,MTBE) and contacting this extract with a suitable base (e.g., KOH).

In certain embodiments of the methods provided herein, the base isselected from potassium hydroxide (KOH), potassium tert-butoxide,potassium carbonate, and triethylamine.

In certain embodiments, the preparation of compound (3) can be carriedby reacting 0.3 M compound (2) in MTBE (e.g., crude extract fromketoreductase reaction mixture) with 0.6 M KOH (or other suitable base)in MTBE solution. This reaction reaches >99% conversion to the epoxideof compound (3) within 5 hours and 99.9% conversion within 8 hours(determined via HPLC).

In certain embodiments, the methods for preparing compounds of Formula(III) of the present disclosure comprise the steps of extracting theenzyme reaction mixture with an organic solvent, and contacting theorganic solvent extract with a base. In certain embodiments, the methodis carried out wherein said step of contacting the compound of Formula(II) with base is carried out without first purifying and/or isolatingthe compound of Formula (II) (e.g., a “telescoped” reaction or a“one-pot” reaction).

Thus, in some embodiments, the present disclosure provides in a methodfor preparing a compound of Formula (III) (e.g., compound (3)) the stepof converting the compound of Formula (I) to a compound of Formula (II)(e.g., compound (2)) using a ketoreductase of the present disclosure. Insome embodiments of preparing compounds of Formula (III), the methodfurther comprises the step of contacting the compound of Formula (II)with base. In certain embodiments, the method is carried out whereinsaid step of contacting the compound of Formula (II) with base iscarried out without first purifying and/or isolating the compound ofFormula (II).

In certain embodiments, the method further comprises exchanging (orswapping) the organic solvent of the organic solvent extract with acrystallization solvent, and crystallizing the compound of Formula (III)from the crystallization solvent. In certain embodiments, the organicsolvent extract is MTBE which is exchanged for the crystallizationsolvent, heptane.

Other organic solvents that may be used for extraction andcrystallization according to the methods provided herein are thosewell-known in the art and accessible to the ordinary artisan, includingwell-known hydrocarbons, ethers, esters, and alcohols e.g.,acetonitrile, n-butanol, toluene, isopropyl acetate.

As noted above, any of the ketoreductase polypeptides described herein,including those exemplified in Table 2, can be used in the methods.Moreover, in some embodiments, the methods can use a ketoreductasepolypeptides comprising an amino acid sequence that is at least about70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO:2, and, further, that comprises, ascompared to SEQ ID NO:2, at least one amino acid substitution selectedfrom the group consisting of: the proline residue at position 2 isreplaced with an aliphatic or nonpolar amino acid selected from amongalanine, leucine, valine, isoleucine, glycine and methionine; the valineresidue at position 28, in a conservative change, is replaced with analiphatic or nonpolar amino acid selected from among alanine, leucine,valine, isoleucine, glycine and methionine; the alanine residue atposition 34 is replaced with a polar amino acid selected from amongasparagine, glutamine, serine, and threonine; the alanine residue atposition 47, in a conservative change, is replaced with an aliphatic ornonpolar amino acid selected from among alanine, leucine, valine,isoleucine, glycine and methionine; the glutamic acid residue atposition 50 is replaced with a basic amino acid selected from lysine andarginine; the aspartic acid residue at position 81 is replaced with apolar amino acid selected from among asparagine, glutamine, serine, andthreonine; the serine residue at position 90 is replaced with analiphatic or nonpolar amino acid selected from among alanine, leucine,valine, isoleucine, glycine and methionine; the isoleucine residue atposition 91 is, in a conservative change, replaced with an aliphatic ornonpolar amino acid selected from among alanine, leucine, valine,isoleucine, glycine and methionine, while in other aspects, theisoleucine residue at position 91 is replaced with an aromatic aminoacid selected from among tyrosine, tryptophan, and phenylalanine, or abasic amino acid selected from among lysine and arginine; the lysineresidue at position 94 is, in a conservative change, replaced withanother basic amino acid, arginine; the aspartic acid residue atposition 112 is replaced with an aromatic amino acid selected from amongtyrosine, tryptophan, and phenylalanine; the glycine residue at position117 is replaced with an acidic amino acid selected from among asparticacid and glutamic acid; the serine residue at position 143 is replacedwith a basic amino acid selected from among lysine and arginine; thevaline residue at position 144 is replaced with a cysteine or a polaramino acid selected from among asparagine, glutamine, serine, andthreonine; the glycine residue at position 145, in either a conservativeor non-conservative change, may be replaced with a nonpolar amino acidselected from among alanine, leucine, valine, isoleucine, and methionineor an aliphatic amino acid selected from among alanine, leucine, valine,isoleucine; the arginine residue at position 148 is replaced with aconstrained amino acid selected from among proline and histidine; thealanine residue at position 150, in conservative or non-conservativechange, is replaced with another nonpolar or aliphatic amino acidselected from among leucine, valine, isoleucine, glycine and methionine,or a polar amino acid selected from among asparagine, glutamine, serine,and threonine, or an aromatic amino acid selected from among tyrosine,tryptophan, and phenylalanine; the phenylalanine residue at position 152is replaced with a nonpolar or aliphatic amino acid selected from amongalanine, leucine, valine, isoleucine, glycine and methionine; theasparagine residue at position 153 is replaced with a nonpolar oraliphatic amino acid selected from among alanine, leucine, valine,isoleucine, glycine and methionine, or with a constrained amino acidselected from among histidine and proline; the threonine residue atposition 158, in a conservative change, is replaced with another polaramino acid selected from among asparagine, glutamine, and serine; theglycine residue at position 190, in either a conservative or anonconservative change, is replaced with either a nonpolar or analiphatic amino acid selected from among alanine, valine, leucine,isoleucine, glycine, and methionine, or a polar amino acid selected fromamong asparagine, glutamine, and serine, or a proline; the serineresidue at position 198, in a conservative change, is replaced withanother polar amino acid selected from among asparagine, glutamine, andthreonine; the isoleucine residue at position 199 is, in a conservativechange replaced with another aliphatic or nonpolar amino acid selectedfrom among alanine, leucine, valine, glycine, and methionine, or with apolar amino acid selected from among asparagine, glutamine, serine, andthreonine; the methionine residue at position 200, in a conservativechange, is replaced with another nonpolar amino acid selected from amongalanine, leucine, valine, isoleucine, and glycine; the valine atposition 204, in a non-conservative change, is replaced with an aromaticamino acid selected from among tyrosine, tryptophan, and phenylalanine;the alanine residue at position 217 is replaced with a polar amino acidselected from among asparagine, glutamine, serine and threonine; theisoleucine residue at position 225, in a conservative change, isreplaced with an other nonpolar amino acid selected from among valine,leucine, glycine, and methionine; the proline residue at position 231 isreplaced with an aromatic amino acid selected from among tyrosine,tryptophan, and phenylalanine; the alanine residue at position 232, in aconservative change, is replaced with another nonpolar amino acidselected from among leucine, isoleucine, valine, glycine, andmethionine; the glutamic acid residue at position 233 is replaced with apolar amino acid selected from among asparagine, glutamine, serine, andthreonine; the aspartic acid residue at position 244 is replaced with anonpolar amino acid selected from among alanine, leucine, isoleucine,valine, glycine, and methionine; the phenylalanine residue at position260, in a conservative change, is replaced with another aromatic aminoacid selected from among tyrosine and tryptophan; and the serine residueat position 261, in a conservative change, is replaced with anotherpolar amino acid selected from among asparagine, glutamine, andthreonine. The forgoing improved ketoreductase polypeptides may furthercomprise additional modifications, including substitutions, deletions,insertions, or combinations thereof. The substitutions can benon-conservative substitutions, conservative substitutions, or acombination of non-conservative and conservative substitutions. In someembodiments, these ketoreductase polypeptides can have optionally fromabout 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40mutations at other amino acid residues. In some embodiments, the numberof modifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15,16, 18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.

In some embodiments, the methods can use an improved ketoreductasepolypeptide of the present disclosure that comprises an amino acidsequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, andthat further comprises, as compared to SEQ ID NO:2, at least one aminoacid substitution selected from the group consisting of: the prolineresidue at position 2 is replaced with leucine (P2L); the valine residueat position 28 is replaced with alanine (V28A); the alanine residue atposition 34 is replaced with serine (A34S); the alanine residue atposition 47 is replaced with valine (A47V); the glutamic acid residue atposition 50 is replaced with lysine (E50K); the aspartic acid residue atposition 81 is replaced with asparagine (D81N); the serine residue atposition 90 is replaced with valine (590V); the isoleucine residue atposition 91 is replaced with an amino acid selected from among leucine(I91L), tryptophan (I91W), arginine (I91R), and lysine (I91K); thelysine residue at position 94 is replaced with arginine (K94R); theaspartic acid residue at position 112 is replaced with tyrosine (D112Y);the glycine residue at position 117 is replaced with aspartic acid(G117D); the serine residue at position 143 is replaced with arginine(S143R); the valine residue at position 144 is replaced with an aminoacid selected from among cysteine (V144C) and threonine (V144T); theglycine residue at position 145 is replaced with an amino acid selectedfrom among alanine (G145A) and valine (G145V); the arginine residue atposition 148 is replaced with histidine (R148H); the alanine residue atposition 150 is replaced with an amino acid selected from among glycine(A150G), isoleucine (A150I), serine (A150S), and tryptophan (A150W); thephenylalanine residue at position 152 is replaced with leucine (F152L);the asparagine residue at position 153 is replaced with an amino acidselected from among glycine (N153G), valine (N153V), and histidine(N153H); the threonine residue at position 158 is replaced with serine(T158S); the glycine residue at position 190 is replaced with an aminoacid selected from among alanine (G190A), proline (G190P), glutamine(G190Q), and valine (G190V); the serine residue at position 198 isreplaced with asparagine (S198N); the isoleucine residue at position 199is replaced with an amino acid selected from among glycine (I199G),methionine (I199M), leucine (I199L), and asparagine (I199N); themethionine residue at position 200 is replaced with isoleucine (M200I);the valine residue at position 204 is replaced with phenylalanine(V204F); the alanine residue at position 217 is replaced with threonine(A217T); the isoleucine residue at position 225 is replaced with valine(I225V); the proline residue at position 231 is replaced withphenylalanine (P231F); the alanine residue at position 232 is replacedwith valine (A232V); the glutamic acid residue at position 233 isreplaced with glutamine (E233Q); the aspartic acid residue at position244 is replaced with glycine (D244G); the phenylalanine residue atposition 260 is replaced with tyrosine (F260Y); and the serine residueat position 261 is replaced with asparagine (S261N). In certainembodiments, a ketoreductase polypeptide of the present disclosurecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 66, 68, 70,72, 74, 76, 78, and 80. The forgoing improved ketoreductase polypeptidesmay further comprise additional modifications, including substitutions,deletions, insertions, or combinations thereof. The substitutions can benon-conservative substitutions, conservative substitutions, or acombination of non-conservative and conservative substitutions. In someembodiments, these ketoreductase polypeptides can have optionally fromabout 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14,1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-25, 1-30, 1-35 or about 1-40mutations at other amino acid residues. In some embodiments, the numberof modifications can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15,16, 18, 20, 22, 24, 26, 30, 35 or about 40 other amino acid residues.

In some embodiments, the methods of the present disclosure use aketoreductase comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,65, 66, 68, 70, 72, 74, 76, 78, 80, and combinations thereof. Exemplarygroups comprising combinations of sequences include: the groupconsisting of SEQ ID NOs 4, 6, 14, 16, 18, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 50, 52, 54, and 56; the group consisting of SEQ ID NOs 6,18, 22, 30, 38, 40, 50, 52, 54, and 56; and the group consisting of SEQID NOs 6, 50, 52, and 56.

In some embodiments of the method, the product has greater than about90%, 95%, 97%, 98%, 99%, or even greater diastereomeric excess of theN-protected (2R,3S)-3-amino-1-chloro-4-phenylbutan-2-ol, wherein theketoreductase polypeptide comprises an amino acid sequence correspondingto SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, and 58.

In some embodiments of the method, at least about 45% of a racemicsubstrate mixture is reduced to the product in less than 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 hours when themethod is conducted with greater than or equal to 200 g/L substrate andless than or equal to 2 g/L ketoreductase enzyme (but more than 0 g/Lenzyme), wherein the ketoreductase polypeptide comprises an amino acidsequence corresponding to SEQ ID NO:6, 50, 52, and 56.

As is known by those of skill in the art, ketoreductase-catalyzedreduction reactions typically require a cofactor. Reduction reactionscatalyzed by the engineered ketoreductase enzymes described herein alsotypically require a cofactor, although many embodiments of theengineered ketoreductases require far less cofactor than reactionscatalyzed with wild-type ketoreductase enzymes. As used herein, the term“cofactor” refers to a non-protein compound that operates in combinationwith a ketoreductase enzyme. Cofactors suitable for use with theengineered ketoreductase enzymes described herein include, but are notlimited to, NADP⁺ (nicotinamide adenine dinucleotide phosphate), NADPH(the reduced form of NADP⁺), NAD⁺ (nicotinamide adenine dinucleotide)and NADH (the reduced form of NAD⁺). Generally, the reduced form of thecofactor is added to the reaction mixture. The reduced NAD(P)H form canbe optionally regenerated from the oxidized NAD(P)⁺ form using acofactor regeneration system.

The term “cofactor regeneration system” refers to a set of reactantsthat participate in a reaction that reduces the oxidized form of thecofactor (e.g., NADP⁺ to NADPH). Cofactors oxidized by theketoreductase-catalyzed reduction of the keto substrate are regeneratedin reduced form by the cofactor regeneration system. Cofactorregeneration systems comprise a stoichiometric reductant that is asource of reducing hydrogen equivalents and is capable of reducing theoxidized form of the cofactor. The cofactor regeneration system mayfurther comprise a catalyst, for example an enzyme that catalyzes thereduction of the oxidized form of the cofactor by the reductant.Cofactor regeneration systems to regenerate NADH or NADPH from NAD+ orNADP+, respectively, are known in the art and may be used in the methodsdescribed herein.

Suitable exemplary cofactor regeneration systems that may be employedinclude, but are not limited to, glucose and glucose dehydrogenase,formate and formate dehydrogenase, glucose-6-phosphate andglucose-6-phosphate dehydrogenase, a secondary (e.g., isopropanol)alcohol and secondary alcohol dehydrogenase, phosphite and phosphitedehydrogenase, molecular hydrogen and hydrogenase, and the like. Thesesystems may be used in combination with either NADP⁺/NADPH or NAD⁺/NADHas the cofactor. Electrochemical regeneration using hydrogenase may alsobe used as a cofactor regeneration system. See, e.g., U.S. Pat. Nos.5,538,867 and 6,495,023, both of which are incorporated herein byreference. Chemical cofactor regeneration systems comprising a metalcatalyst and a reducing agent (for example, molecular hydrogen orformate) are also suitable. See, e.g., PCT publication WO 2000/053731,which is incorporated herein by reference.

The terms “glucose dehydrogenase” and “GDH” are used interchangeablyherein to refer to an NAD+ or NADP+-dependent enzyme that catalyzes theconversion of D-glucose and NAD+ or NADP+ to gluconic acid and NADH orNADPH, respectively. Equation (1), below, describes the glucosedehydrogenase-catalyzed reduction of NAD+ or NADP+ by glucose:

Glucose dehydrogenases that are suitable for use in the practice of themethods described herein include both naturally occurring glucosedehydrogenases, as well as non-naturally occurring glucosedehydrogenases. Naturally occurring glucose dehydrogenase encoding geneshave been reported in the literature. For example, the Bacillus subtilis61297 GDH gene was expressed in E. coli and was reported to exhibit thesame physicochemical properties as the enzyme produced in its nativehost (Vasantha et al., 1983, Proc. Natl. Acad. Sci. USA 80:785). Thegene sequence of the B. subtilis GDH gene, which corresponds to GenbankAcc. No. M12276, was reported by Lampel et al., 1986, J. Bacteriol.166:238-243, and in corrected form by Yamane et al., 1996, Microbiology142:3047-3056 as Genbank Acc. No. D50453. Naturally occurring GDH genesalso include those that encode the GDH from B. cereus ATCC 14579(Nature, 2003, 423:87-91; Genbank Acc. No. AE017013) and B. megaterium(Eur. J. Biochem., 1988, 174:485-490, Genbank Acc. No. X12370; J.Ferment. Bioeng., 1990, 70:363-369, Genbank Acc. No. GI216270). Glucosedehydrogenases from Bacillus sp. are provided in PCT publication WO2005/018579 as SEQ ID NOS: 10 and 12 (encoded by polynucleotidesequences corresponding to SEQ ID NOS: 9 and 11, respectively, of thePCT publication), the disclosure of which is incorporated herein byreference.

Non-naturally occurring glucose dehydrogenases may be generated usingknown methods, such as, for example, mutagenesis, directed evolution,and the like. GDH enzymes having suitable activity, whether naturallyoccurring or non-naturally occurring, may be readily identified usingthe assay described in Example 4 of PCT publication WO 2005/018579, thedisclosure of which is incorporated herein by reference. Exemplarynon-naturally occurring glucose dehydrogenases are provided in PCTpublication WO 2005/018579 as SEQ ID NO: 62, 64, 66, 68, 122, 124, and126. The polynucleotide sequences that encode them are provided in PCTpublication WO 2005/018579 as SEQ ID NO: 61, 63, 65, 67, 121, 123, and125, respectively. All of these sequences are incorporated herein byreference. Additional non-naturally occurring glucose dehydrogenasesthat are suitable for use in the ketoreductase-catalyzed reductionreactions disclosed herein are provided in U.S. application publicationNos. 2005/0095619 and 2005/0153417, the disclosures of which areincorporated herein by reference.

Glucose dehydrogenases employed in the ketoreductase-catalyzed reductionreactions described herein may exhibit an activity of at least about 10μmol/min/mg and sometimes at least about 10² μmol/min/mg or about 10³μmol/min/mg, up to about 10⁴ μmol/min/mg or higher in the assaydescribed in Example 4 of PCT publication WO 2005/018579.

The ketoreductase-catalyzed reduction reactions described herein aregenerally carried out in a solvent. Suitable solvents include water,organic solvents (e.g., ethyl acetate, butyl acetate, 2-propanol(isopropanol or IPA), 1-octanol, heptane, octane, methyl t-butyl ether(MTBE), toluene, and the like), ionic liquids (e.g.,1-ethyl-4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, and the like). In someembodiments, aqueous solvents, including water and aqueous co-solventsystems, are used.

Exemplary aqueous co-solvent systems have water, pH buffering salts, andone or more organic solvent. In general, an organic solvent component ofan aqueous co-solvent system is selected such that it does notcompletely inactivate the ketoreductase enzyme. Appropriate co-solventsystems can be readily identified by measuring the enzymatic activity ofthe specified engineered ketoreductase enzyme with a defined substrateof interest in the candidate solvent system, utilizing an enzymeactivity assay, such as those described herein.

The organic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Generally, when an aqueous co-solventsystem is employed, it is selected to be biphasic, with water dispersedin an organic solvent, or vice-versa. Generally, when an aqueousco-solvent system is utilized, it is desirable to select an organicsolvent that can be readily separated from the aqueous phase. Ingeneral, the ratio of water to organic solvent in the co-solvent systemis typically in the range of from about 90:10 to about 10:90 (v/v)organic solvent to water, and between 80:20 and 20:80 (v/v) organicsolvent to water. The co-solvent system may be pre-formed prior toaddition to the reaction mixture, or it may be formed in situ in thereaction vessel. In certain embodiments, the aqueous co-solvent systemcomprises isopropyl alcohol (IPA) of about 5%-40% (v/v), about 5%-20%(v/v), about 10-20% (v/v), about 15% (v/v), or about 10% (v/v).

The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. Generally, the reduction can be carried outat a pH of about 10 or below, usually in the range of from about 5 toabout 10. In some embodiments, the reduction is carried out at a pH ofabout 9.5 or below, usually in the range of from about 6.5 to about 9.5.The reduction may be carried out at a pH of about 7.0 to about 9.5. Incertain embodiments, the reduction is carried out at a pH of about 8.5to about 9.5. In a particular embodiment, the reduction is carried outat a pH of about 9.0. Alternatively, the reduction may be carried out atneutral pH, i.e., about 7.

In certain embodiments where an aqueous co-solvent is used, the reactionconditions for reduction can comprise a pH of about 8.5 to about 9.5 andabout 5% to about 40% IPA, about pH 9.0 to 9.5 and about 25% to about40% IPA, about pH 8.5 to 9.5 and about 5% to about 15% IPA, or about pH9.0 and about 10% IPA. In certain embodiments, the reaction conditionsfor reduction can comprise a pH of about 6.5 to about 7.0 and about 5%to about 15% IPA, or about pH 7.0 and about 5% to 10% IPA.

During the course of the reduction reactions, the pH of the reactionmixture (e.g., the aqueous co-solvent solution) may change. The pH ofthe reaction mixture may be maintained at a desired pH or within adesired pH range by the addition of an acid or a base during the courseof the reaction. Alternatively, the pH may be controlled by using anaqueous solvent that comprises a buffer. Suitable buffers to maintaindesired pH ranges are known in the art and include, for example,phosphate buffer, triethanolamine buffer (TEA), and the like.Combinations of buffering and acid or base addition may also be used.Accordingly, in certain embodiments, the aqueous co-solvents having pHand organic solvent (e.g., IPA) in certain ranges specified above, mayalso comprise a buffer such as TEA.

When the glucose/glucose dehydrogenase cofactor regeneration system isemployed, the co-production of gluconic acid (pKa=3.6), as representedin equation (1) causes the pH of the reaction mixture to drop if theresulting aqueous gluconic acid is not otherwise neutralized. The pH ofthe reaction mixture may be maintained at the desired level by standardbuffering techniques, wherein the buffer neutralizes the gluconic acidup to the buffering capacity provided, or by the addition of a baseconcurrent with the course of the conversion. Combinations of bufferingand base addition may also be used. Suitable buffers to maintain desiredpH ranges are described above. Suitable bases for neutralization ofgluconic acid are organic bases, for example amines, alkoxides and thelike, and inorganic bases, for example, hydroxide salts (e.g., NaOH),carbonate salts (e.g., NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basicphosphate salts (e.g., K₂HPO₄, Na₃PO₄), and the like. The addition of abase concurrent with the course of the conversion may be done manuallywhile monitoring the reaction mixture pH or, more conveniently, by usingan automatic titrator as a pH stat. A combination of partial bufferingcapacity and base addition can also be used for process control.

When base addition is employed to neutralize gluconic acid releasedduring a ketoreductase-catalyzed reduction reaction, the progress of theconversion may be monitored by the amount of base added to maintain thepH. Typically, bases added to unbuffered or partially buffered reactionmixtures over the course of the reduction are added in aqueoussolutions.

In some embodiments, the co-factor regenerating system can comprises aformate dehydrogenase. The terms “formate dehydrogenase” and “FDH” areused interchangeably herein to refer to an NAD+ or NADP+-dependentenzyme that catalyzes the conversion of formate and NAD+ or NADP+ tocarbon dioxide and NADH or NADPH, respectively. Formate dehydrogenasesthat are suitable for use as cofactor regenerating systems in theketoreductase-catalyzed reduction reactions described herein includeboth naturally occurring formate dehydrogenases, as well asnon-naturally occurring formate dehydrogenases. Formate dehydrogenasesinclude those corresponding to SEQ ID NOS: 70 (Pseudomonas sp.) and 72(Candida boidinii) of PCT publication WO 2005/018579, which are encodedby polynucleotide sequences corresponding to SEQ ID NOS: 69 and 71,respectively, of PCT publication 2005/018579, the disclosures of whichare incorporated herein by reference. Formate dehydrogenases employed inthe methods described herein, whether naturally occurring ornon-naturally occurring, may exhibit an activity of at least about 1μmol/min/mg, sometimes at least about 10 μmol/min/mg, or at least about10² μmol/min/mg, up to about 10³ μmol/min/mg or higher, and can bereadily screened for activity in the assay described in Example 4 of PCTpublication WO 2005/018579.

As used herein, the term “formate” refers to formate anion (HCO₂ ⁻),formic acid (HCO₂H), and mixtures thereof. Formate may be provided inthe form of a salt, typically an alkali or ammonium salt (for example,HCO₂Na, KHCO₂NH₄, and the like), in the form of formic acid, typicallyaqueous formic acid, or mixtures thereof. Formic acid is a moderateacid. In aqueous solutions within several pH units of its pKa(pK_(a)=3.7 in water) formate is present as both HCO₂ ⁻ and HCO₂H inequilibrium concentrations. At pH values above about pH 4, formate ispredominantly present as HCO₂ ⁻. When formate is provided as formicacid, the reaction mixture is typically buffered or made less acidic byadding a base to provide the desired pH, typically of about pH 5 orabove. Suitable bases for neutralization of formic acid include, but arenot limited to, organic bases, for example amines, alkoxides and thelike, and inorganic bases, for example, hydroxide salts (e.g., NaOH),carbonate salts (e.g., NaHCO₃), bicarbonate salts (e.g., K₂CO₃), basicphosphate salts (e.g., K₂HPO₄, Na₃PO₄), and the like.

For pH values above about pH 5, at which formate is predominantlypresent as HCO₂ ⁻, Equation (2) below, describes the formatedehydrogenase-catalyzed reduction of NAD+ or NADP+ by formate.

When formate and formate dehydrogenase are employed as the cofactorregeneration system, the pH of the reaction mixture may be maintained atthe desired level by standard buffering techniques, wherein the bufferreleases protons up to the buffering capacity provided, or by theaddition of an acid concurrent with the course of the conversion.Suitable acids to add during the course of the reaction to maintain thepH include organic acids, for example carboxylic acids, sulfonic acids,phosphonic acids, and the like, mineral acids, for example hydrohalicacids (such as hydrochloric acid), sulfuric acid, phosphoric acid, andthe like, acidic salts, for example dihydrogenphosphate salts (e.g.,KH₂PO₄), bisulfate salts (e.g., NaHSO₄) and the like. Some embodimentsutilize formic acid, whereby both the formate concentration and the pHof the solution are maintained.

When acid addition is employed to maintain the pH during a reductionreaction using the formate/formate dehydrogenase cofactor regenerationsystem, the progress of the conversion may be monitored by the amount ofacid added to maintain the pH. Typically, acids added to unbuffered orpartially buffered reaction mixtures over the course of conversion areadded in aqueous solutions.

The terms “secondary alcohol dehydrogenase” and “sADH” are usedinterchangeably herein to refer to an NAD+ or NADP+-dependent enzymethat catalyzes the conversion of a secondary alcohol and NAD+ or NADP+to a ketone and NADH or NADPH, respectively. Equation (3), below,describes the reduction of NAD+ or NADP+ by a secondary alcohol,illustrated by isopropanol.

Secondary alcohol dehydrogenases that are suitable for use as cofactorregenerating systems in the ketoreductase-catalyzed reduction reactionsdescribed herein include both naturally occurring secondary alcoholdehydrogenases, as well as non-naturally occurring secondary alcoholdehydrogenases. Naturally occurring secondary alcohol dehydrogenasesinclude known alcohol dehydrogenases from, Thermoanaerobium brockii,Rhodococcus etythropolis, Lactobacillus kefiri, Lactobacillus brevis,Lactobacillus minor, Novosphingobium aromaticivorans and non-naturallyoccurring secondary alcohol dehydrogenases include engineered alcoholdehdyrogenases derived therefrom. Secondary alcohol dehydrogenasesemployed in the methods described herein, whether naturally occurring ornon-naturally occurring, may exhibit an activity of at least about 1μmol/min/mg, sometimes at least about 10 μmol/min/mg, or at least about10² μmol/min/mg, up to about 10³ μmol/min/mg or higher.

Suitable secondary alcohols include lower secondary alkanols andaryl-alkyl carbinols. Examples of lower secondary alcohols includeisopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol, 3-pentanol,3,3-dimethyl-2-butanol, and the like. In one embodiment the secondaryalcohol is isopropanol. Suitable aryl-alkyl carbinols includeunsubstituted and substituted 1-arylethanols.

When a secondary alcohol and secondary alcohol dehydrogenase areemployed as the cofactor regeneration system, the resulting NAD+ orNADP+ is reduced by the coupled oxidation of the secondary alcohol tothe ketone by the secondary alcohol dehydrogenase. Some engineeredketoreductases also have activity to dehydrogenate a secondary alcoholreductant. In some embodiments using secondary alcohol as reductant, theengineered ketoreductase and the secondary alcohol dehydrogenase are thesame enzyme. Therefore, in certain embodiments, the reactions of thepresent disclosure are as depicted in Schemes 6 and 7, below:

In carrying out embodiments of the ketoreductase-catalyzed reductionreactions described herein employing a cofactor regeneration system,e.g. as depicted in Schemes 6 and 7, the reactions may be run under lowpressure and/or increased temperature to effect removal of thepropan-2-one product. Such embodiments may further comprise the additionof isopropanol as the reaction proceeds, e.g., by continuous feeding orbatch additions.

In carrying out embodiments of the ketoreductase-catalyzed reductionreactions described herein employing a cofactor regeneration system,either the oxidized or reduced form of the cofactor may be providedinitially. As described above, the cofactor regeneration system convertsoxidized cofactor to its reduced form, which is then utilized in thereduction of the ketoreductase substrate.

In some embodiments, cofactor regeneration systems are not used. Forreduction reactions carried out without the use of a cofactorregenerating systems, the cofactor is added to the reaction mixture inreduced form.

In some embodiments, when the process is carried out using whole cellsof the host organism, the whole cell may natively provide the cofactor.Alternatively or in combination, the cell may natively or recombinantlyprovide the glucose dehydrogenase.

In carrying out the stereospecific reduction reactions described herein,the engineered ketoreductase enzyme, and any enzymes comprising theoptional cofactor regeneration system, may be added to the reactionmixture in the form of the purified enzymes, whole cells transformedwith gene(s) encoding the enzymes, and/or cell extracts and/or lysatesof such cells. The gene(s) encoding the engineered ketoreductase enzymeand the optional cofactor regeneration enzymes can be transformed intohost cells separately or together into the same host cell. For example,in some embodiments one set of host cells can be transformed withgene(s) encoding the engineered ketoreductase enzyme and another set canbe transformed with gene(s) encoding the cofactor regeneration enzymes.Both sets of transformed cells can be utilized together in the reactionmixture in the form of whole cells, or in the form of lysates orextracts derived therefrom. In other embodiments, a host cell can betransformed with gene(s) encoding both the engineered ketoreductaseenzyme and the cofactor regeneration enzymes.

Whole cells transformed with gene(s) encoding the engineeredketoreductase enzyme and/or the optional cofactor regeneration enzymes,or cell extracts and/or lysates thereof, may be employed in a variety ofdifferent forms, including solid (e.g., lyophilized, spray-dried, andthe like) or semisolid (e.g., a crude paste).

The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like, followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde or immobilization to asolid phase (e.g., Eupergit C, and the like).

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a pre-chilled lyophilizationchamber, followed by the application of a vacuum. After the removal ofwater from the samples, the temperature is typically raised to 4° C. fortwo hours before release of the vacuum and retrieval of the lyophilizedsamples.

The quantities of reactants used in the reduction reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of ketoreductase substrate employed.

The following guidelines can be used to determine the amounts ofketoreductase, cofactor, and optional cofactor regeneration system touse. Generally, keto substrates can be employed at a concentration ofabout 20 g/L to 300 g/L using from about 50 mg/L to about 5 g/L ofketoreductase and about 10 mg to about 150 mg of cofactor. Those havingordinary skill in the art will readily understand how to vary thesequantities to tailor them to the desired level of productivity and scaleof production. Appropriate quantities of optional cofactor regenerationsystem may be readily determined by routine experimentation based on theamount of cofactor and/or ketoreductase utilized. In general, thereductant (e.g., glucose, formate, isopropanol) is utilized at levelsabove the equimolar level of ketoreductase substrate to achieveessentially complete or near complete conversion of the ketoreductasesubstrate.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points. For example, the cofactor regeneration system,cofactor, ketoreductase, and ketoreductase substrate may be added firstto the solvent.

For improved mixing efficiency when an aqueous co-solvent system isused, the cofactor regeneration system, ketoreductase, and cofactor maybe added and mixed into the aqueous phase first. The organic phase maythen be added and mixed in, followed by addition of the ketoreductasesubstrate. Alternatively, the ketoreductase substrate may be premixed inthe organic phase, prior to addition to the aqueous phase.

Suitable conditions for carrying out the ketoreductase-catalyzedreduction reactions described herein include a wide variety ofconditions which can be readily optimized by routine experimentationthat includes, but is not limited to, contacting the engineeredketoreductase enzyme and substrate at an experimental pH and temperatureand detecting product, for example, using the methods described in theExamples provided herein.

The ketoreductase catalyzed reduction is typically carried out at atemperature within the range of from about 15° C. to about 85° C., fromabout 20° C. to about 80° C., from about 25° C. to about 75° C., fromabout 30° C. to about 70° C., from about 35° C. to about 65° C., fromabout 40° C. to about 60° C., or from about 45° C. to about 55° C. Incertain embodiments, the ketoreductase catalyzed reduction is carriedout at a temperature of about 45° C.

The reduction reaction is generally allowed to proceed until essentiallycomplete, or nearly complete, conversion of substrate to product isobtained. The reduction of substrate to product can be monitored usingknown methods by detecting substrate and/or product. Suitable methodsinclude gas chromatography, HPLC, and the like. Conversion yields of thealcohol reduction product generated in the reaction mixture aregenerally greater than about 50%, may also be greater than about 60%,may also be greater than about 70%, may also be greater than about 80%,may also be greater than 90%, and are often greater than about 97%, 98%,or even 99%.

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

6. EXAMPLES Example 1 Wild-Type Ketoreductase Gene Acquisition andConstruction of Expression Vectors

The ketoreductase (KRED) encoding gene from wild-type Novosphingobiumaromaticivorans (SEQ ID NO:2) was designed for expression in E. coliusing standard codon optimization. (Standard codon-optimization softwareis reviewed in e.g., “OPTIMIZER: a web server for optimizing the codonusage of DNA sequences,” Puigbò et al., Nucleic Acids Res. 2007 July;35(Web Server issue): W126-31. Epub 2007 Apr. 16.) Genes weresynthesized using oligonucleotides composed of 42 nucleotides and clonedinto expression vector pCK110900, which is depicted as FIG. 3 in USPatent Application Publication 20060195947, which is hereby incorporatedby reference herein, under the control of a lac promoter. The expressionvector also contained the P15a origin of replication and thechloramphenicol resistance gene. Resulting plasmids were transformedinto E. coli W3110 or E. coli BL21 using standard methods.

Polynucleotides encoding the engineered ketoreductase polypeptides ofthe present disclosure were also cloned into vector pCK110900 forexpression in E. coli W3110 or E. coli BL21. Multiple rounds of directedevolution of the codon-optimized KRED gene were carried out yielding thevariant sequences listed in Table 2.

Example 2 Shake-Flask Procedure for Production of KetoreductasePolypeptides

A single microbial colony of E. coli containing a plasmid encoding anengineered ketoreductase of interest was inoculated into 50 mL LuriaBertani broth containing 30 μg/ml chloramphenicol and 1% glucose. Cellswere grown overnight (at least 16 hours) in an incubator at 30° C. withshaking at 250 rpm. The culture was diluted into 250 ml Terrific Broth(12 g/L bacto-tryptone, 24 g/L yeast extract, 4 ml/L glycerol, 65 mMpotassium phosphate, pH 7.0, 1 mM MgSO₄) containing 30μg/mlchloramphenicol, in a 1 liter flask to an optical density at 600 nm(OD600) of 0.2 and allowed to grow at 30° C. Expression of theketoreductase gene was induced by addition ofisopropyl-β-D-thiogalactoside (“IPTG”) to a final concentration of 1 mMwhen the OD600 of the culture is 0.6 to 0.8 and incubation was thencontinued overnight (at least 16 hours).

Cells were harvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet was resuspended with an equalvolume of cold (4° C.) 100 mM triethanolamine (chloride) buffer, pH 7.0(optionally including 2 mM MgSO₄), and harvested by centrifugation asabove. The washed cells were resuspended in two volumes of the coldtriethanolamine (chloride) buffer and passed through a French Presstwice at 12,000 psi while maintained at 4° C. Cell debris was removed bycentrifugation (9000 rpm, 45 minutes, 4° C.). The clear lysatesupernatant was collected and stored at −20° C. Lyophilization of frozenclear lysate provides a dry shake-flask powder of crude ketoreductasepolypeptide. Alternatively, the cell pellet (before or after washing)can be stored at 4° C. or −80° C.

Example 3 Fermentation Procedure for Production of KetoreductasePolypeptides

Bench-scale fermentations were carried out at 30° C. in an aerated,agitated 15 L fermentor using 6.0 L of growth medium (0.88 g/L ammoniumsulfate, 0.98 g/L of sodium citrate; 12.5 g/L of dipotassium hydrogenphosphate trihydrate, 6.25 g/L of potassium dihydrogen phosphate, 6.2g/L of Tastone-154 yeast extract, 0.083 g/L ferric ammonium citrate, and8.3 ml/L of a trace element solution containing 2 g/L of calciumchloride dihydrate, 2.2 g/L of zinc sulfate septahydrate, 0.5 g/Lmanganese sulfate monohydrate, 1 g/L cuprous sulfate heptahydrate, 0.1g/L ammonium molybdate tetrahydrate and 0.02 g/L sodium tetraborate).The fermentor was inoculated with a late exponential culture of E. coliW3110 or E. coli BL21 containing the plasmid encoding the engineeredketoreductase gene of interest (grown in a shake flask as described inExample 2) to a starting OD600 of 0.5 to 2.0. The fermentor was agitatedat 500-1500 rpm with air supplied to the fermentation vessel at 1.0-15.0L/minutes to maintain a dissolved oxygen level of 30% saturation orgreater. The pH of the culture was maintained at 7.0 by addition of 20%v/v ammonium hydroxide. Growth of the culture was maintained by additionof a feed solution containing 500 g/L cerelose, 12 g/L ammonium chlorideand 10.4 g/L magnesium sulfate heptahydrate. After the culture reachedan OD600 of 50, expression of ketoreductase was induced by addition ofisopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mMand fermentation continued for another 14 hours. The culture was thenchilled to 4° C. and maintained at that temperature until harvested.Cells were collected by centrifugation at 5000 G for 40 minutes at 4° C.Harvested cells were used directly in the following downstream recoveryprocess or were stored at 4° C. or frozen at −80° C. until such use.

The cell pellet was resuspended in 2 volumes of 100 mM triethanolamine(chloride) buffer, pH 6.8, at 4° C. to each volume of wet cell paste.The intracellular ketoreductase was released from the cells by passingthe suspension through a homogenizer fitted with a two-stagehomogenizing valve assembly using a pressure of 12000 psig. The cellhomogenate was cooled to 4° C. immediately after disruption. A solutionof 10% w/v polyethyleneimine, pH 7.2, was added to the lysate to a finalconcentration of 0.5% w/v and stirred for 30 minutes. The resultingsuspension was clarified by centrifugation at 5000G in a standardlaboratory centrifuge for 30 minutes. The clear supernatant was decantedand concentrated ten-fold using a cellulose ultrafiltration membranewith a molecular weight cut off of 30 kD. The final concentrate wasdispensed into shallow containers, frozen at −20° C. and lyophilized toa powder. The crude ketoreductase polypeptide powder is stored at −80°C.

Example 4 Determination of Percent Conversion and Diastereomeric Purityfor Ketoreductase Catalyzed Reduction of Compound (1) ((S)-tert-butyl4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate) to the correspondingalcohol, Compound (2) (tert-butyl(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate)

The conversion rate ketoreductase catalyzed stereoselective reduction ofcompound (1) ((S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate)to the corresponding alcohol, compound (2) (tert-butyl(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate) was determinedby sampling enzymatic reaction mixtures at time intervals (e.g., athours 0.5, 2, 4, 7, 9, and 24) using an Agilent 1200 HPLC equipped withan Agilent XDB C18 (15 cm length, 4.6 mm diameter), using 60% MeCN, 40%water (isocratic) as eluent at a flow 1.8 ml/min; and a columntemperature 25° C. Compound (1) retention time: 3.119 minutes; compound(2) retention time: 2.378 minutes. The amounts of substrate (compound(1)) and product (compound (2)) were determined based HPLC peak areasdetected at 210 nm.

Diastereopurity of the product of the ketoreductase polypeptidecatalyzed reaction, compound (2) (tert-butyl(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate) was determinedusing an Agilent 1200 HPLC equipped with Agilent XDB C18 (15 cm length,4.6 mm diameter) using 50% MeCN, 50% water (isocratic) as the eluent ata flow rate of 1.50 mL/min at a temperature of 20° C. Desireddiastereomer of compound (2) retention time: 5.083 min; and undesireddiastereomer (and substrate) retention time: 4.050 min.

Example 5 Prescreen for Engineered Ketoreductase Polypeptides Capable ofReducing Isopropanol in the Presence of NADP to Yield NADPH and Acetone

This example illustrates a prescreening assay used to identify variantgenes encoding ketoreductases capable of reducing isopropanol in thepresence of NADP⁺ to produce acetone and NADPH. An E. coli colonycontaining a plasmid encoding an engineered ketoreductase was pickedusing a Q-Bot® robotic colony picker (Genetix USA, Inc., Boston, Mass.)into 96-well shallow well microtiter plates containing 180 μL TerrificBroth (TB), 1% glucose and 30 μg/mL chloramphenicol (CAM). Cells weregrown overnight at 30° C. with shaking at 200 rpm. A 10 μL aliquot ofthis culture was then transferred into 96-deep well plates containing390 μL Terrific Broth (TB), 1 mM MgSO₄ and 30 μg/mL CAM. Afterincubation of the deep-well plates at 30° C. with shaking at 250 rpm for2 to 3 hours, recombinant gene expression within the cultured cells wasinduced by addition of IPTG to a final concentration of 1 mM. The plateswere then incubated at 30° C. with shaking at 250 rpm for 18 hours.

Cells were pelleted by centrifugation (4000 RPM, 10 minutes, 4° C.),resuspended in 400 μL lysis buffer and lysed by shaking at roomtemperature for 2 hours. The lysis buffer contains 100 mMtriethanolamine (chloride) buffer, pH 7, 1 mg/mL lysozyme, 500 μg/mLpolymixin B sulfate (“PMBS”) and 1 mM MgSO₄. After sealing withaluminum/polypropylene laminate heat seal tape (Velocity 11 (Menlo Park,Calif.), Cat #06643-001), the plates were shaken vigorously for 2 hoursat room temperature. Cell debris was collected by centrifugation (4000RPM, 10 minutes, 4° C.) and the clear supernatant was assayed directlyor stored at 4° C. until use.

In this assay, 20 μl of sample (diluted in 100 mMtriethanolamine(chloride) buffer, at the same pH as the lysis buffer,and 1 mM MgSO₄) was added to 180 μl of an assay mixture in a well of96-well black microtiter plates. Assay buffer consists of 100 mMtriethanolamine (chloride) buffer, pH 7, 50% isopropyl alcohol (IPA), 1mM MgSO₄ and 222 μM NADP⁺. The reaction was followed by measuring thereduction in fluorescence of NADP⁺ as it is converted to NADPH using aFlexstation® instrument (Molecular Devices, Sunnyvale, Calif.). NADPHfluorescence was measured at 445 nm upon excitation at 330 nm. Ifdesired, samples of lysates can be preincubated at 25-40° C. in thepresence or absence of 50% IPA prior to addition to the assay mixture.

Example 6 Screening for Engineered Ketoreductase Polypeptides Capable ofStereoselective Conversion of the Substrate, Compound (1)((S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate) to thecorresponding product, compound (2) (tert-butyl(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate)

The codon-optimized ketoreductase gene of derived from Novosphingobiumaromaticivorans (SEQ ID NO: 1) constructed as in Example 1, wassubmitted to mutagenesis using directed evolution methods describedabove and the population of mutant DNA molecules were transformed into asuitable E. coli host strain. Antibiotic resistant transformants wereselected and processed to identify those expressing a ketoreductasehaving an improved ability to convert compound (1) to compound (2).

Cell selection, growth, induction of the variant ketoreductase genes andcollection of cell pellets were as described in Example 5. Cell pelletswere lysed by addition of 400 μL lysis buffer (1 mM MgSO₄, 0.5 mg/mlpolymyxin B sulfate (“PMBS”), 1 mg/ml lysozyme, 100 mM triethanolamine(pH ˜6), and 1 mg/mL NADP⁺) to each well. The plates were sealed, shakenvigorously for two hours at room temperature, and then centrifuged at4000 rpm for 10 minutes at 4° C. The supernatants was recovered andstored at 4° C. until use.

Enzymatic reduction assay: An aliquot (450 μL) of a mixture ofisopropanol and solid substrate ((S)-tert-butyl4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate) was added to each well of aCostar® deep well plate using a Multidrop instrument (MTX Lab Systems,Vienna Va.), followed by robotic addition of 50 μL of the recoveredlysate supernatant using a Multimek™ instrument (Multimek, Inc., SantaClara Calif.), to provide a reaction comprising 10 mg/ml substrate(S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate, 0.1 mg/mlNADP⁺, 10 mM triethanolamine pH ˜6, and 10% isopropanol (v/v). Theplates were heat sealed with aluminum/polypropylene laminate heat sealtape (Velocity 11 (Menlo Park, Calif.), Cat #06643-001) at 170° C. for2.5 seconds and then shaken overnight (at least 16 hours) at ambienttemperature. Reactions were quenched by the addition of 1 ml ofmethyl-t-butyl ether (MTBE). Plates were resealed, shaken for 5 minutes,and then centrifuged at 4000 rpm for 10 minutes. A 250 μL aliquot of thecleared reaction mixture was transferred to a new shallow wellpolypropylene plate (Costar #3365), which was sealed, after which theextracts are subjected to HPLC analysis using methods described above(e.g., see Example 4).

High-throughput screening assay at pH ˜6 and 10% IPA (v/v): 50 μl ofcell lysate containing 1 g/L NADP⁺ was transferred to a deep well plate(Costar #3960) containing 450 μl of an assay mix (per 100 ml assay mix:5 ml 100 mM triethanolamine (chloride) (pH 7), 13.4 g (S)-tert-butyl4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate, and 10 ml isopropylalcohol). After sealing the plates, reactions were run for at least 16hours at ambient temperature. Reactions were quenched by the addition of1 ml of 95% MTBE, and the plates sealed with aluminum/polypropylenelaminate heat seal tape (Velocity 11 (Menlo Park, Calif.), Cat#06643-001), shaken for 5-10 min, and centrifuged at 4000 rpm for 10minutes. A 250 μL aliquot of the cleared reaction mixture wastransferred to a new shallow well polypropylene plate (Costar #3365),which was then sealed. The extracts prepared in this manner weresubjected to HPLC analysis as described above.

Engineered ketoreductase polypeptides capable of converting(S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate to tert-butyl(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate with highconversion rate (e.g., at least about 70-95% in 24 hours) and highdiastereomeric purity (e.g., at least about 85-99% d.e.) were identifiedusing the procedures disclosed above. Multiple iterations of theseprocedures were carried out in which one or more engineeredketoreductase gene with improved properties were isolated from one roundof mutagenesis and used as the starting material for subsequent roundsof mutagenesis and screening. Some of the improved engineeredketoreductases resulting from these multiple rounds of directedevolution are disclosed herein and listed in Table 2.

Example 7 Stereoselective Reduction of (S)-tert-butyl4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate, compound (1), usingisopropyl alcohol for co-factor regeneration by engineeredketoreductases derived from Novosphingobium aromaticivorans

Engineered ketoreductase enzymes derived from Novosphingobiumaromaticivorans as described above were assayed for use in a preparativescale reduction of (S)-tert-butyl4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate as follows. A 100 μL solutionof the engineered ketoreductase to be tested (10 mg/mL) and NADP-Na (1mg/mL) in 100 mM triethanolamine(chloride) buffer pH 7 were combined ina 5 mL reaction vial equipped with a magnetic stir bar. Subsequently, 85μL of isopropyl alcohol (“IPA”) was added to the enzyme/NADP-Nasolution, followed by 120 mg of compound (1). The reaction was stirredat ambient temperature and conversion of the compound (1) to compound(2) was monitored by HPLC analysis of samples taken periodically over a24 hour period from the reaction using analytical methods disclosed inExample 4.

Table 2 identifies the ketoreductase variants (by polynucleotide andpolypeptide SEQ ID NO), the amino acid mutations relative to wild-typeketoreductase polypeptide of SEQ ID NO:2, and the relative activity ofeach variant, relative to the activity of the wild-type enzyme havingthe amino acid sequence of SEQ ID NO: 2. As shown by the results listedin Table 2, nearly all of the engineered ketoreductases have at least120% (i.e., 1.2-fold or greater) of the activity of the wild-typepolypeptide activity, and several of the engineered ketoreductases(e.g., polypeptides of SEQ ID NOs: 6, 50, 52, 56) have mutationsresulting in improved activity at least 300% greater (i.e., 3-fold orgreater) than that of SEQ ID NO: 2. These results illustrate thatengineered ketoreductases derived from the ketoreductase Novosphingobiumaromaticivorans disclosed herein provide improved activities compared tothe wild-type ketoreductase of SEQ ID NO:2 for the reduction ofcompounds of Formula (I) such as compound (1).

Example 8 Use of Engineered Ketoreductases in a StereoselectivePreparative Scale Conversion of Compound (1) to Compound (2), andCompound (2) to Compound (3)

A 1 L jacketed process reactor, equipped with over head stirrer, baffleand internal thermometer was charged sequentially with 90.0 g ofcompound (1), 400 ml of a 100 mM triethanolamine solution (pH 9.0), 60ml IPA and NAD⁺ (300 mg). The resulting slurry was stirred for 10 minand 600 mg of the engineered ketoreductase polypeptide of SEQ ID NO: 6was added. The reaction mixture was heated to 45° C. and stirred at 150rpm for the first 4 hours and at 250 rpm afterwards. In-process HPLCanalysis to determine conversion of compound (1) to compound (2) wascarried out on the reaction (as described in Example 4). Afterin-process analysis indicated 99.8% conversion (at 9 hours) the reactionwas cooled to 20° C.

MTBE (600 mL) was added to the reaction slurry and agitated at 250 rpmfor 50 min. Phases were allowed to separate and the aqueous layerremoved. The MTBE phase was collected separately. The aqueous layer wasrecharged and MTBE (300 mL) added. The biphasic mixture was agitated for250 rpm for 45 min. The phases were allowed to separate and the aqueouslayer removed. HPLC analysis of the aqueous phase (as described fordetermining conversion in Example 4) indicated that >99% of the producthad been removed. The combined MTBE layers were filtered through a padof celite (30 g), the filter cake was washed with 90 mL MTBE, and theunified MTBE phases washed with 90 mL water at 250 rpm for 15 min. Thephases were allowed to separate and the aqueous phase removed. Thepurity of the desired product compound (2) was determined to be 98.4%according to HPLC.

KOH (39.7 g, 85% w/w) was added to the organic phase containing compound(2) and stirred at 250 rpm and 25° C. After in-process HPLC analysisindicated >99.9% conversion (at 8 hours) 180 ml of water was added andthe biphasic mixture stirred for 30 min at 250 rpm. The phases wereallowed to separate and the aqueous phase removed. The wash was repeatedtwice with water (90 mL and 180 mL). The remaining MTBE phase (1 L) wasconcentrated to 400 mL and then 600 ml of n-heptane was added. Theresulting mixture was concentrated again to 400 mL using a jackettemperature of 50° C. while incrementally reducing the pressure to 105Torr. This procedure was repeated once. GC analysis of the remainingn-heptane layer indicates that <0.7% MTBE remained and n-heptane wasadded to give an overall volume of 1 L. The solution was stirred at 120rpm and the temperature of the solution adjusted to 20° C. The solutionwas seeded with 20 mg of pure compound (3) and stirred for 1 hour. Thetemperature was gradually reduced in 0.5° C. steps over 150 min to 17.5°C. After 1 hour of additional stirring crystal formation was observed.The resulting more viscous solution was stirred at 400 rpm and thetemperature reduced to 0° C. and stirred for 30 min. The reactor wasdrained and the white mass filtered under reduced pressure, washed withcold n-heptane (2×180 ml) and dried at approx 20 mm Hg for 24 hours.This provided 64.4 g (81% yield) of compound (3),tert-butyl(S)-1-((R)-oxiran-2-yl)-2-phenylethylcarbamate, in a singlecrop as a white solid with chemical purity of 98.9% and diastereomericpurity of >99.9% de. The balance of the yield was in the mother liquorsand could be isolated as a second crop to provide a near quantitativetotal yield of approximately 98-99%. It is reasonable to expect that amodified crystallization process can lead to nearly quantitative singlecrop yields of the pure product of compound (3).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered ketoreductase polypeptide capableof converting (S)-tert-butyl 4-chloro-3-oxo-1-phenylbutan-2-ylcarbamateto tert-butyl (2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamatewith a relative activity at least 1.2-fold that of the polypeptide ofSEQ ID NO:2, wherein the engineered ketoreductase polypeptide comprisesan amino acid sequence at least 90% identical to SEQ ID NO:2 andincludes one or more amino acid substitutions relative to SEQ ID NO: 2,selected from the group consisting of P2L; V28A; A34S; A47V; E50K; D81N;590V; I191L; I91W; I91R; I91K; K94R; D112Y; G117D; S143R; V144T; G145A;R148H; A150G; F152L; N153G; N153V; N153H; T1585; G190A; S198N; I199M;I199L; I199N; M200I; A217T; I225V; P231F; A232V; E233Q; D244G; F260Y;and S261N.
 2. The engineered ketoreductase polypeptide of claim 1,wherein 4 the polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:6, 4, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 50, 52, 54, 56, 60, 62, 65, 66, 68, 70, 72, 74,76, 78, and
 80. 3. The engineered ketoreductase polypeptide of claim 1,wherein the relative activity is at least 1.5-fold that of thepolypeptide of SEQ ID NO: 2 and the engineered ketoreductase polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 6, 18, 22, 30, 38, 40, 50, 52, 54, and
 56. 4. The engineeredketoreductase polypeptide of claim 1, wherein the amino acid sequence ofsaid engineered ketoreductase polypeptide comprises at least one of thefollowing substitutions: I91L; I91W; I91R; I91K; V144T; G145A; A150G;N153G; N153V; N153H; G190A; I199M; I199L; I199N; and F260Y, wherein thesubstitutions are relative to SEQ ID NO:2.
 5. The engineeredketoreductase polypeptide of claim 1, wherein the amino acid sequence ofsaid engineered ketoreductase polypeptide comprises at least one of thefollowing substitutions: I91R; G145A; N153G; N153V; N153H; G190A; andF260Y, wherein the substitutions are relative to SEQ ID NO:2.
 6. Theengineered ketoreductase polypeptide of claim 4, wherein the relativeactivity is at least 3-fold that of the polypeptide of SEQ ID NO:2, andwherein the engineered ketoreductase polypeptide sequence comprises thesubstitution G145A, wherein the sequence is relative to SEQ ID NO:2. 7.The engineered ketoreductase polypeptide of claim 1, wherein thepolypeptide is capable of converting a reaction mixture comprising aninitial concentration of at least 10 g/L (S)-tert-butyl4-chloro-3-oxo-1-phenylbutan-2-ylcarbamate to tert-butyl(2S,3R)-4-chloro-3-hydroxy-1-phenylbutan-2-ylcarbamate in at least 97%diastereomeric excess with a conversion rate of at least 70% in 24hours.
 8. A composition comprising the polypeptide of claim
 1. 9. Aprocess for preparing a compound of Formula (II) in greater than 95%diastereomeric excess, said process comprising:

contacting a compound of Formula (I)

with the engineered ketoreductase polypeptide of claim 1 under suitablereaction conditions.
 10. The process of claim 9, wherein R₁ is anitrogen protecting group selected from the group consisting of: formyl,trityl, methoxytrityl, tosyl, phthalimido, acetyl, trichloroacetyl,chloroacetyl, bromoacetyl, iodoacetyl, benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (FMOC), 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), trihaloacetyl, benzyl, benzoyl, andnitrophenylacetyl.
 11. The process of claim 9, wherein R₁ ist-butoxycarbonyl.
 12. The process of claim 9, wherein the reactionconditions comprise one or more of: (a) a pH of about 6.5 to about 9.5;(b) a temperature of from about 25° C. to about 60° C.; (c) an aqueousco-solvent system comprising an organic solvent to water ratio fromabout 90:10 (v/v) to about 10:90 (v/v); an aqueous co-solvent systemcomprising from about 5% to about 40% isopropanol; (e) the engineeredketoreductase polypeptide concentration is less than 5 g/L; and/or thecompound of Formula (I) concentration is at least 100 g/L.
 13. Theprocess of claim 9, wherein the reaction conditions comprise one or moreof: (a) a pH of about 9.0; (b) a temperature of about 45° C.; (c) anaqueous co-solvent system comprising about 10% isopropanol; (d) theengineered ketoreductase polypeptide concentration is less than 1 g/L;and/or (e) the compound of Formula (I) concentration is at least 150g/L.
 14. The process of claim 9, further comprising a cofactorregenerating system selected from the group consisting of glucosedehydrogenase and glucose, formate dehydrogenase and formate, phosphitedehydrogenase and phosphite, and isopropanol and a secondary alcoholdehydrogenase.
 15. The process of claim 14, wherein the cofactorregenerating system is a secondary alcohol dehydrogenase that is theengineered ketoreductase polypeptide.
 16. The process of claim 9,wherein at least 95% of the compound of Formula (I) is converted to thecompound of Formula (II) in less than 24 hours.
 17. The process of claim9, wherein at least 95% of the compound of Formula (I) is converted tothe compound of Formula (II) in less than 24 hours, wherein the compoundof Formula (I) concentration is at least 150 g/L and the engineeredketoreductase polypeptide concentration is less than 1 g/L.
 18. Aprocess for preparing a compound of Formula (III) in greater than about95% diastereomeric excess, said process comprising:

(a) contacting a compound of Formula (I) with the engineeredketoreductase polypeptide of claim 1 under suitable reaction conditions,

thereby forming a reaction mixture comprising a compound of Formula (II)

(b) extracting the reaction mixture with an organic solvent; and (c)contacting the organic solvent extract with a base to produce a compoundof Formula (III).
 19. The process of claim 18, wherein R₁ is a nitrogenprotecting group selected from the group consisting of: formyl, trityl,methoxytrityl, tosyl, phthalimido, acetyl, trichloroacetyl,chloroacetyl, bromoacetyl, iodoacetyl, benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (FMOC), 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), trihaloacetyl, benzyl, benzoyl, andnitrophenylacetyl.
 20. The process of claim 18, wherein said step ofcontacting the compound of Formula (II) with base is carried out withoutpurifying and/or isolating the compound of Formula (II).
 21. The processof claim 18, further comprising crystallizing the compound of Formula(III) from the organic solvent extract.
 22. The process of claim 18,wherein the organic solvent is methyl t-butyl ether (MTBE) and furthercomprising exchanging the organic solvent extract with heptane andcrystallizing the compound of Formula (III) from the heptane.